Registration Dossier

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Administrative data

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Effects on fertility

Description of key information

Short description of key information:

A screening for reproductive toxicity study (OECD 421) was performed in Wistar rats dosed by oral gavage at 0,100, 200 and 400 mg/kg b.w. Dosing started 2 weeks prior to mating, and continued in both sexes during the mating period. Males were further dosed after the mating period until the minimum dosing period of 28 days was completed. Dosing of confirmed females was continued throughout the pregnancy and including day 3 post partum. Based on the above findings, toxicity signs in animals treated with high dose (400 mg/kg b.w): hunched posture, abnormal gait, tremor, respiratory distress, dullness, salivation, pilo-erection, test substance related adverse effect on body weight changes and on most of the reproduction parameters, statistically decrease in absolute weight of testis and epididymis in high doses group. It is concluded that the dose 100 and 200 mg/kg b.w of N-n-butylbenzene sulphonamide was considered to be safe and non-toxic, while 400 mg/kg b.w of N-n-butylbenzenesulphonamide was considered toxic to Wistar rats in terms of parental and reproduction effects, therefore the NOAEL of the test substance was regarded as 200 mg/kg b.w.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A screening for reproductive toxicity study (OECD 421) was performed in Wistar rats dosed by oral gavage at 0,100, 200 and 400 mg/kg b.w. (IIBAT, 2007). Dosing started 2 weeks prior to mating, and continued in both sexes during the mating period. Males were further dosed after the mating period until the minimum dosing period of 28days was completed. Dosing of confirmed females was continued throughout the pregnancy and including day 3 post partum. Based on the above findings, toxicity signs in animals treated with high dose (400 mg/kg b.w): hunched posture, abnormal gait, tremor, respiratory distress, dullness, salivation, pilo-erection, test substance related adverse effects on body weight changes and on reproduction parameters, statistically significant decreases in absolute weight of testis and epididymis in high doses group. It is concluded that the dose 100 and 200 mg/kg b.w of N-n-butylbenzene sulphonamide was considered to be safe and non-toxic, while 400 mg/kg b.w of N-n-butylbenzenesulphonamide was considered toxic to Wistar rats in terms of parental and reproduction effects, therefore the NOAEL of the test substance was regarded as 200 mg/kg b.w.

Effects on developmental toxicity

Description of key information

N-butylbenzenesulphonamide administered to pregnant Hannover Wistar rats by oral gavage at dose levels of 12, 40 and 120 mg/kg bw/day, daily from gestation days GD 6 to 19, was associated with following effects: at 120 mg/kg bw/day, maternal toxicity was observed with decreased body weight gain (15 and 30% for absolute and net mean values, respectively), decreased food consumption, and foetal toxicity evident as a reduction in mean foetal body weight (approximately 9%). In two litters, mild to moderate dilatation of chorionic vessels was observed microscopically, which correspond with enlargement of placenta. At 40 mg/kg bw/day, there were no effects considered adverse or test item related in the dams or at the external, visceral and skeletal examination of the foetuses. The No Observed Adverse Effect Level (NOAEL) under the conditions of this study for maternal toxicity was found to be 40 mg/kg bw/day and for foetal developmental toxicity 120 mg/kg bw/day.

A supporting DRF (dose range finder) (Test Facility Reference No. 20175108) was conducted to select dose levels for the Prenatal Developmental Toxicity Study in rabbits (Test Facility Study No. 20175109). No guidelines were applicable as this study was intended for dose level selection purposes only.

Six female rabbits per group received doses of 1250, 2500 or 5000 ppm (Approx. 37, 67 or 131 mg test item/kg body weight).

Each viable fetus of animals surviving to planned necropsy was externally examined in detail and weighed. Nonviable fetuses of animals surviving to planned necropsy (the degree of autolysis is minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed. Sex was not determined. No visceral (internal) or skeletal examination was performed.

Based on the results of the DRF (dose range finder), it was concluded that a dose level of 5000 ppm did induce an effect on body weight and food consumption. Values recovered to normal levels by the end of treatment and no secondary effects were noted on fetal development, exposure to 5000 ppm was therefore considered not to induce adverse effects on maternal or developmental parameters and was considered insufficient as highest dose in the main study.

However, as it could not be excluded that the effect on food consumption and body weight would increase and induce excessive toxicity at higher ppm levels, concentrations higher than 7500 ppm were considered inappropriate for a study in pregnant rabbits.

Selected dose levels for the main study were 1900, 3800 and 7500 ppm.

In an OECD Guideline 414 (Prenatal Developmental Toxicity Study) the potential of N-butylbenzenesulphonamide to induce developmental toxicity was determined after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given via diet to time-mated female New Zealand White Rabbits from Days 6 to 29 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 1900, 3800 and 7500 ppm (0, 54, 105, 195 mg/kg bw/day).

Based on the results in this prenatal developmental toxicity study, no adverse effects were observed for developmental, maternal toxicity and teratogenicity.

Based on the results, a maternal and developmental No Observed Adverse Effect Level (NOAEL) for N butylbenzenesulphonamide of 7500 ppm (highest dose tested; corresponding to an actual test item intake of 195 mg/kg/day) was established.


Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-05-13 until 2014-06-06 (in vivo)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Hannover Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony
- Age at study initiation: Young adult female rats, nulliparous and non-pregnant, approximately 11 weeks old
- Weight at onset of treatment: Not exceeding ± 20% of the mean weight, and was in the range of 180-222 g.
- Housing: Standard laboratory conditions; individual housing. Type II and/or III polycarbonate cages with Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). The bedding material was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Diet (e.g. ad libitum): The animals were provided with ssniff® SM R/M “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (Ssniff Spezialdiäten GmbH, D-59494 Soest Germany) ad libitum.
- Water (e.g. ad libitum): The animals were provided with tap water as for human consumption, in water bottles, ad libitum. The quality control analysis of the water is performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary).
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 – 24.6°C
- Humidity (%):33 - 66%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 May 2014 To: 6 June 2014

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (PEG 400) at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd.
Doses of 12, 40 and 120 mg/kg bw/day were selected for the study.
The oral route is a possible route of exposure to the test item in humans and is considered suitable to provide the systemic exposure required on this developmental toxicology study.
A constant volume of 1 mL/kg bw was administered to animals in all dose groups, including the controls. The individual volume of the treatment was based on the most recent individual body weights.
The control or test item dose formulations were administered to mated, sperm positive assumed pregnant female rats daily by oral gavage on a 7 days/week basis, approximately at similar times with minor variations as practical, from Gestation Day (GD) 6 to GD19.
Formulations were prepared fresh prior to administration to animals or at 2-4-day intervals and were used according to stability results.
Stability of N-butylbenzenesulphonamide in the vehicle (PEG 400) was assessed in the conditions employed on the study (concentration range and storage conditions of the dose formulations pending use, according to CiToxLAB Hungary study code 14/103-316AN). According to the results, the test item is stable in vehicle in concentration range of 1 mg/mL - 400 mg/mL for 24 hours at room temperature (RT, 15-30°C) and for up to seven days when stored refrigerated at 2-8°C.
Analysis of formulations (concentration measurements) was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Duplicate samples were taken from the test item formulations twice during the study (during the first and last weeks of treatment). Similarly, one sample (duplicate) was taken on each occasion from the Group 1 (control) solution for concentration measurements.
All formulations were found to be in the range of 101 to 104% of nominal concentration. No test item was detected in the control samples.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Based on the results of the pilot developmental study (CiToxLAB study code: 14/103-105PE) and as confirmed in accordance with the analytical method development, polyethylene glycol (PEG 400) was selected as vehicle.
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required): BCBK9981V
- Purity:

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulations (concentration measurements) was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Duplicate samples were taken from the test item formulations twice during the study (during the first and last weeks of treatment). Similarly, one sample (duplicate) was taken on each occasion from the Group 1 (control) solution for concentration measurements.

All formulations were found to be in the range of 101 to 104% of nominal concentration. No test item was detected in the control samples.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male: 1-3 females
- Length of cohabitation: approximately 2 hours
- No replacement of first male by another male needed.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (GD0).
On GD13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).
Duration of treatment / exposure:
from gestation day 6 to gestation day 19
Frequency of treatment:
daily on a 7 days/week basis
Duration of test:
Caesarean section and necropsy at gestation day 20
No. of animals per sex per dose:
102 female animals, plus an appropriate number of spares: 25-26 mated female animals/group, 4 groups (one control and 3 test item-treated groups); 24-25 pregnant female animals/group (with implantation sites at necropsy);
60 male animals for mating; no study-procedures were carried out on the male animals; untreated, proven breeders from CiToxLAB Hungary Ltd. spare colony were used.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were set by the Sponsor in consultation with the Study Director based on available data from the previous studies, including the results of a pilot developmental study in the rat (CiToxLAB study code 14/103-105PE, entitled: “N-Butylbenzenesulfonamide (NBBS): A Dose Range Finding Toxicity Study Following Oral (Gavage) Administration in Pregnant Hannover Wistar Rat”), with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.
In the pilot study, marked maternal toxicity, evident in reduced body weight gain and food consumption was observed at 350 and 200 mg/kg bw/day, in addition to the markedly decreased foetal body weight. At 100 mg/kg bw/day, slightly lower body weight gain was observed in dams, (overall mean value by approximately 12%, and corrected body weight by 8%, when compared to control mean), with the mean foetal weight lower than control by approximately 13%.
Based on the above results, doses of 12, 40 and 120 mg/kg bw/day were selected for the study.
The oral route is a possible route of exposure to the test item in humans and is considered suitable to provide the systemic exposure required on this developmental toxicology study.
- Rationale for animal assignment (if not random):
The sperm-positive, assumed pregnant females were allocated to each experimental group (on each mating day) in such a way that the group averages of the body weight were as similar as possible. Females paired with the same male were allocated to different groups on the same mating day.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). Cage-side clinical observations were made at least daily after the treatment.
The animals were monitored for any changes including pertinent behavioural changes and signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical signs were noted during the study.

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: Detailed clinical observations were made on all animals at the onset of treatment (GD6) then weekly.

BODY WEIGHT: Yes
Time schedule for examinations: The body weight of each animal was recorded with a precision of ±1 g on GD 0, 3, 6, 7, 8, 9, 10, 12, 14, 16, 18 and 20.

FOOD CONSUMPTION: Yes
Food was measured with a precision of ±1 g on GD0, 3, 6, 7, 8, 9, 10, 12, 14, 16, 18 and 20. Food consumption was calculated for each interval, including GD6-20 and GD0-20.

POST-MORTEM EXAMINATIONS: Yes
Sacrifice on gestation day 20
Organs examined: The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes. The ovaries and uterus were removed and the pregnancy status ascertained.

OTHER: On GD13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation). The placentas were examined macroscopically, with the exception of few placentas (5 placentas of 4510 litter and one of 4519 litter).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The degree of resorption was described in order to estimate the relative time of death of the conceptus.
Fetal examinations:
- External examinations: Yes: all per litter (live foetuses)
- Soft tissue examinations: Yes: half per litter (visceral examination)
- Skeletal examinations: Yes: half per litter (other half of the litter)
- Head examinations: No data
Statistics:
The statistical evaluation of data was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) by an appropriate statistical method (Bartlett, ANOVA and Duncan, Kruskal-Wallis and Mann-Whitney U tests, Chi2). The homogeneity of variance between groups was checked by Bartlett`s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was made. If the obtained result was significant Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Significant results with inter-group comparisons were further compared using Kruskal-Wallis, and Mann-Whitney U-tests.

The following data were excluded from statistical analysis:
- sperm positive but non pregnant females (total exclusion) [No. 118 (12 mg/kg), 216 (40 mg/kg) and 108 (120 mg/kg)]
- females with ≤ 5 implantation sites (total exclusion) [No. 3525, 4 implantation sites (40 mg/kg) and No. 4525, 4 implantation sites (120 mg/kg) ]

The limit for growth retarded foetuses was calculated from the average body weight of the vehicle control foetuses. A foetus was considered as growth retarded if the deviation from the mean control values was greater than minus two fold standard deviation of all control foetuses. In the present study, this cut-off value was set at 2.632 g.
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Body weight gain of dams at 120 mg/kg bw/day was lower than control mean during the treatment period (by 15 and 30% for absolute and net mean values, respectively) and was accompanied by slightly decreased food consumption.

Details on maternal toxic effects:
There was no unscheduled mortality during the study.

There were no toxicologically significant clinical signs noted following administration of test item.
Immediately after the treatment, minimal salivation and excessive digging of bedding were occasionally observed at 120 mg/kg bw/day from Day 14. These observations were regarded to be procedure related.

Thin fur (focal) or focal alopecia were occasionally observed in 2/24 and 1/24 animals in the Mid and in and High dose groups, respectively (3520 Days 10-20 on dorsal area, 3504 Days 14-20 on left flank; 4515 Days 19-20 ventral thorax). Small skin lesions (scar and crust or local red coloured skin) were also occasionally observed with similar low incidence in the control, low and high dose groups (1/25, 1/24 and 1/24, respectively). These observations were regarded as incidental and not related to the test item.

Compared to the control mean, the body weight of dams at 120 mg/kg bw/day was slightly lower from Day 12 up to Day 20 (by 2.5% - 4.0%). The differences were not statistically significant.

Body weight gains of dams at 120 mg/kg bw/day dose groups were lower than control on GD6-8 (by 54-71%) up to GD12 (by 10-24%) and between GD14-20 (by 8-13%). The differences were statistically significant on GD6-7 (p<0.01) and GD7-8 and 16-18 (p<0.05).

These lower mean values were corresponding with slightly decreased food consumption. The mean body weight gain of treated dams, evaluated for the entire treatment period GD 6-20, was lower than control mean (by 15%), and the difference was statistically significant (p<0.01).

Compared to the control mean, the “corrected” body weight gain (body weight gain between GD0-20 adjusted for the gravid uterine weight) of the dams at 120 mg/kg bw/day was lower by 16%; the net body weight gain during the treatment period (body weight gain between GD6-20 adjusted for the gravid uterine weight) of the dams at 120 mg/kg bw/day was lower by 30%. The differences attained statistical significance (p<0.05 and p<0.01, respectively).

Body weight of dams at 12 and 40 mg/kg bw/day was comparable to the control mean during the treatment period. Although slightly lower body weight gain was noted at 40 mg/kg bw/day between GD6-7 (p<0.05) and GD8-9 (not statistically significant), it was transient and did not result in any differences in overall values including net body weight gains.

It should be noted, that in the Low and Mid groups, the mean body weight gains were slightly lower than control prior to the treatment period (both groups GD0-3 and the Low dose group GD 3-6) and this finding was ascribed to the individual variability of the animals.

The food consumption of dams at 120 mg/kg bw/day was lower, than control, by 8.1% (GD6-20). The difference attained statistical significance (p<0.01).
The more pronounced difference was noted at the beginning of the treatment period (approximately 9-12%). On the following days, the average food consumption was lower, than control by 6.4-9.2%. Except days GD9-10, all differences were statistically significant (p<0.01 and p<0.05).

The food consumption of dams at 12 and 40 mg/kg bw/day groups was did not differ significantly from the control throughout the treatment period. Although statistically significant differences were noted at both groups for several periods, the differences were contributable to the relatively high individual food consumptions of control dams (1511 and 1524).

At necropsy of dams (GD20), no test item related macroscopic changes were found, except few large placentae.
Key result
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: At 120 mg/kg bw/day foetal toxicity evident as a reduction in mean foetal body weight (approximately 9%). In two litters mild to moderate dilatation of chorionic vessels was observed microscopically, which correspond with enlargement of placenta.

Details on embryotoxic / teratogenic effects:
The mean number of corpora lutea and the mean number of implantation sites were comparable with the controls in all treated groups.
The pre-implantation loss was at control level in all treated groups.

The total intrauterine mortality was comparable with the control.

The early embryonic loss did not differ significantly from the control in the test item treated groups.

The late embryonic loss was slightly higher, than control at 120 mg/kg bw/day (with incidence of 4/249 and 1/246 in the high dose and control group. The differences were not statistically significant. Although the causative role of the test item cannot be excluded, since it occurred at the dose level with evidence of maternal toxicity, the incidence of the observation was within the historical control range.

The mean number of viable foetuses was comparable with the control mean.

The sex distribution of foetuses did not differ significantly between the control and treatment groups both for mean and absolute numbers. However, more females than males were observed in the test item treated groups. The difference was of low magnitude, was not statistically significant, nor dose dependent, therefore it was considered to be coincidental.

At 120 mg/kg bw/day, large placentas were recorded in 2/24 litters. In 4519/6 female foetus had large placenta (weight of 1.2g). In 4510 litter all placentas (5/5) were enlarged with weight ranging between 0.93-1.45g.
Weight of randomly selected control placentas was in the normal range.

At histopathology examination, mild to moderate dilatation of chorionic vessels was observed in all examined placentas from the High dose group.

There were no toxicological significant differences in the placenta at evaluation of the 12 and 40 mg/kg bw/day treated groups.

In one litter at 40 mg/kg bw/day (3508/10 female foetus), an excess in the amount of amniotic fluid (polyhydramnions) was noted.

The weight of foetuses at 120 mg/kg bw/day was decreased, by approximately 9% evaluated by both litter mean and group mean when compared to the controls. The differences attained statistical significance (p<0.01). No significant differences in body weight were observed between male and female foetuses at the same dose level.

The incidence of body weight retarded foetuses was similar in the control and High dose groups.

There was no foetal external malformation ascribed to the test item administration.
There were no foetal visceral abnormalities ascribed to the test item administration. The number and percentages of the minor structural changes (variations) recorded in the test item treated groups did not differ significantly from the control group. The variations included for example short brachiocephalic trunk, thymic cord, small renal papilla and small kidney.
These variations occurred with low incidence across the groups and were similar with the historical control data and were considered incidental, unrelated to test item administration.

One malformed foetus was found during the visceral examination at 40 mg/kg bw/day (3508/10 female foetus). In this foetus, narrow oesophagus was noted and the finding was corresponding with polyhydramnions (an excess in the amount of amniotic fluid).
Based on the isolated occurrence, this observation was considered incidental, ascribed to individual variability and not related to treatment, however concurrent control data did not contain this observation.

No skeletal abnormalities that could be correlated with test item administration under the conditions of this study were noted at any of dose levels. The number and percentages of the abnormalities recorded in the test item treated groups was comparable to the control group.
At 120 mg/kg bw/day, no skeletally malformed foetus was found. See overview Table 8.

Minor changes (variations) occurred, including for example in sternal ossification, asymmetric ribs, fused to sternum, dumbbell shaped and/or asymmetric, bipartite vertebral bodies or 3.5 or less metatarsals, observed with low incidence throughout all groups including controls, or with lower incidence in the treated groups than in the controls.

Compared to controls, asymmetric and/or bipartite ossification of the sternum was observed with higher incidence in the test item treated groups (3 in the Low and Mid dose and 5 in the High dose groups versus 1 in control). The differences were of low magnitude and were without attaining statistical significance. Generally, this delayed ossification was limited to sternebrae 5 and 6 and taking into account incidence of all type of altered sternal ossification, it was similar across the groups. Therefore it was considered incidental or background/individual change and not an adverse effect of toxicological significance.

Two skeletally malformed foetuses were found during the study, one in the Mid dose and one in the Low dose group.
At 40 mg/kg bw/day, malformation of the ribs (intercostal and detached rib) was found in one foetus (3522/4, body weight 2.82g). Although the Historical Control data did not contain this malformation, based on the isolated occurrence (1/111) and in the Mid dose group, this observation was considered incidental.
At 12 mg/kg bw/day, in foetus 2524/3 multiple malformation of vertebrae was found. This malformation has been observed in control Wistar rats in this laboratory.
Key result
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: foetal toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1. Maternal and Developmental toxicity study with N-butylbenzenesulphonamide (BBSA) in rats

Parameter

Group:

1

2

3

4

 

 

Dose mg/kg:

0

12

40

120

 

Maternal findings

 

 

 

 

 

 

Treated No. of dams

incidence

25

25

26

26

 

Mortality

incidence

0

0

0

0

 

Clinical observations

 

 

 

 

 

 

  excessive digging (GD14-19)

incidence

0

0

0

17

 

  salivaton, min. (GD14-19)

incidence

0

0

0

23

 

Body weight d 20

g (mean)

311,0

308,9

304,6

298,5

NS

 

% vs Control

100

-0,7

-2,1

-4,0

 

Corrected body weight GD20

g (mean)

256,4

250,9

251,4

246,5

NS

 

% vs Control

100

-2,1

-1,9

-3,9

 

Body weight gain GD6-20

g (mean)

89,6

89,3

83,7

76,3**

DN

 

% vs Control

100

-0,3

-6,6

-15

 

Net body weight gain GD6-20

g (mean)

34,9

31,3

30,5

24,3**

DN

% vs Control

100

-10

-13

-30

 

Food consumption d6-20

g

229

21,8*(1)

21,5*(1)

21,0**

DN

 

% vs Control

100

-4,8

-6,1

-8,1

 

Macroscopic findings

 

 

 

 

 

 

  large placenta

litter incid.

0

0

0

2

 

 

fetal incid.

0

0

0

6

 

Reproductive mother

findings

 

 

 

 

 

 

 No. mated

incidence

25

25

26

26

 

 No. pregnant

incidence

25

24

25

25

NS

 No. with litters

incidence

25

24

25

25

NS

 No. wiht<5 implantations

incidence

0

0

1

1

NS

 No. Observed

incidence

25

24

24

24

NS

 Pregnancy index

Index

100

96

96,15

96,15

NS

 Gestation index

Index

100

100

100

100

NS

 Early delivery

incidence

0

0

0

0

NS

 Death

incidence

0

0

0

0

NS

 Accidental kill

incidence

0

0

0

0

NS

 Total resorptions

incidence

0

0

0

0

NS

 Mean corpora lutea

Mean/group

11,1

11,8

11,6

12,0

NS

 Mean implantations

Mean/group

9,8

10,5

10,1

10,4

NS

 Pre-implantation loss

%

11,3

10,2

12, 7

12,6

NS

 Post-implantation loss

%

6,8

4,7

7,4

8,4

NS

  Early embryonic loss

%

6,4

4,4

6,5

6,8

NS

  Late embryonic loss

%

0,4

0,3

0,8

1,6

NS

  Total intrauterine mortality

%

17,0

14,5

19,0

19,8

NS

Litter findings

 

 

 

 

 

 

 Mean live fetuses

Mean/group

9,2

10,0

9,3

9,5

NS

 Sex ratio

M%

47,1

43,9

41,1

43,1

NS

 Mean pup weight (/litter)

g

3,338

3,429

3,325

3,031**

DN

 

% vs Control

100

+2,7

-0,4

-9,2

 

Malformations

 

 

 

 

 

  External

incidence

0/231

0/240

0/224

0/228

NS

  Visceral

incidence

0/116

0/120

1/113

0/116

NS

    oesophagus narrow

 

 

 

1

 

 

  Skeletal

incidence

0/115

1/120

1/111

0/112

NS

    multiple malf. vertebrae

 

 

1

 

 

 

    Intercostal &

detached rib

 

 

 

1

 

 

Variations - developmental

incidences

 

 

 

 

 

  External

incidence

0/231

0/240

0/224

0/228

NS

  Visceral

incidence

5/116

8/120

11/113

9/116

NS

  Skeletal

incidence

22/115

16/120

24/111

26/112

NS

  Retarded in body weight

incidence

13

3**

9

11

CH

 

Legend
CH = Chi Squaire Test
DN = Duncan’s Multiple Range Test;
GD: Gestation day
NS = not significant
(1) Statistical significance attributable to high individual food consumption of
 2 control dams

Conclusions:
The No Observed Adverse Effect Level (NOAEL) under the conditions of this study for maternal toxicity was found to be 40 mg/kg bw/day and for foetal developmental toxicity 120 mg/kg bw/day.
Executive summary:

A developmental toxicity study was performed to assess the effects of the test item N-butylbenzenesulphonamide on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy. The dams (one control and 3-treated groups) were treated daily by oral (gavage) administration, from gestation day GD6 up to and including GD19. Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20.The dose levels were 12, 40 and 120 mg/kg bw/day. Control dams were treated with vehicle polyethylene glycol (PEG 400) only.

 

The number of confirmed pregnant, evaluated dams in the dose groups treated at 12, 40 and 120 mg/kg bw/day was 24 in each of group and 25 in the control group. There was no unscheduled mortality during the study. There were no toxicologically significant clinical signs noted following administration of test item. Body weight gain of dams at 120 mg/kg bw/day was lower than control mean during the treatment period (by 15 and 30% for absolute and net mean values, respectively) and was accompanied by slightly decreased food consumption.

 

No test item related macroscopic changes were found at necropsy of dams on GD20, except few large placentae at 120 mg/kg bw/day (6 foetal and 2 litter incidences, respectively). Mild to moderate dilatation of chorionic vessels was observed in all examined placentas from the High dose group.

 

There were no test-item related significant differences between the treated and control animals regarding the pre-implantation loss or early embryonic death, post-implantation loss, or total intrauterine mortality. The mean number of viable foetuses was comparable with the control mean. The sex distribution of foetuses did not differ significantly from the controls. The weight of foetuses at 120 mg/kg bw/day was decreased by approximately 9% evaluated by both litter mean and group mean when compared to the controls. The differences attained statistical significance. No significant differences in body weight were observed between male and female foetuses at the same dose level.

 

There were no foetal external, visceral or skeletal abnormalities ascribed to the test item administration.

 

Two malformed foetuses were found at 40 mg/kg bw/day, one visceral (narrow oesophagus with polyhydramnions) and one skeletal (intercostal and detached rib). Although the Historical Control data did not contain these malformations, based on the isolated occurrence (1/113 and 1/111) in the Mid dose group, this observation was considered incidental. At 12 mg/kg bw/day, one foetus with multiple malformation of vertebrae was found. This malformation has been observed in control Wistar rats in this laboratory.

The No Observed Adverse Effect Level (NOAEL) under the conditions of this study for maternal toxicity was found to be 40 mg/kg bw/day and for foetal developmental toxicity 120 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2014-03-18 until 2014-04-16 (in vivo)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
no guideline followed
Principles of method if other than guideline:
As a preliminary study, this study did not follow strictly a specific guideline, but principles from OECD Guideline no. 414 and OECD Guidance no. 43, and was designed to allow selection of appropriate dose levels for use in the forthcoming prenatal developmental toxicity study.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Hannover Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony
- Age at study initiation: Young adult female rats, nulliparous and non-pregnant, approximately 12 weeks old at initiation of treatment
- Weight at study initiation: Did not exceed ± 20% of the mean weight at onset of treatment and were in the range of 202-247 g
- Housing: Standard laboratory conditions during the study. Type II and/or III polycarbonate cages.
- Diet (e.g. ad libitum): The animals were provided with ssniff® SM R/M-Z+H “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (ssniff Spezialdiäten GmbH, D-59494 Soest Germany) ad libitum.
- Water (e.g. ad libitum): The animals were provided with tap water as for human consumption, ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 – 25.0°C
- Humidity (%):34 - 48%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 March 2014 To: 16 April 2014
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle, polyethylene glycol (PEG 400) at appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd.
The oral route is a possible route of exposure to the test item in humans and is considered suitable to provide the systemic exposure required on this preliminary dose range finding developmental toxicology study.
A constant volume of 1 mL/kg body weight was administered to all dose groups, including the controls. The individual volume of the treatment was based on the most recent individual body weight of the animals.
The control or test item dose formulations were administered to mated, sperm positive assumed pregnant female rats daily by oral gavage on a 7 days/week basis, approximately at similar times as practical, from GD5 to GD19.
Formulations were prepared at 3-7days intervals and were used according to stability assessment results within 7 days while stored refrigerated.
Stability of N-butylbenzenesulfonamide (NBBS) in the vehicle (PEG 400) was assessed in the conditions employed on the study (concentration range and storage conditions of the dose formulations pending use, according to CiToxLAB Hungary study code 14/103-316AN). According to the results, the test item was stable in vehicle in concentration range of 1 mg/mL - 400 mg/mL for 24 hours at room temperature (RT, 15-30°C) and for up to seven days when stored refrigerated at 2-8°C.
Analysis of test item formulations for concentration was performed using a validated HPLC-UV method in the Analytical Laboratory of CiToxLAB Hungary Ltd. Duplicate samples were taken from test item formulations once (during the first week of treatment).
One set was analyzed and one set was retained as a back-up, for possible confirmatory analysis, which was not required. Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements.
The test item concentration in the dose formulations was determined once during the treatment period (during the first week of treatment). No test item was detected in the control solution samples. All formulations were found to be in the range of 98 to 100% of nominal concentrations.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Based on the results of the preliminary formulation trial, polyethylene glycol (PEG 400) was selected as vehicle, suitable for the study purposes.
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required): BCBK9981V
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the dose formulations was determined once during the treatment period (during the first week of treatment). No test item was detected in the control solution samples. All formulations were found to be in the range of 98 to 100% of nominal concentrations. The test item formulations were considered acceptable
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male: 3 females
- Length of cohabitation: approximately 2 hours
- No replacement of first male by another male needed.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (GD0).
On GD13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).
Duration of treatment / exposure:
from gestation day 5 to gestation day 19
Frequency of treatment:
daily on a 7 days/week basis
Duration of test:
Caesarean section and necropsy at gestation day 20
No. of animals per sex per dose:
29 females, plus an appropriate number of spares: 6-7 mated females/group in 3 test item-treated groups and 9 mated females in control group)
25 males were used for mating; no study-procedures were carried out on males; untreated, proven breeders from CiToxLAB Hungary Ltd. spare colony were used.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were set by the Sponsor in consultation with the Study Director based on available data. According to the information provided by the Sponsor, mortality occurred at 400 mg/kg bw/day (oral gavage) in the female rat during a developmental screening study (OECD 421), therefore the high dose is set at 350 mg/kg/bw/day.

The oral route is a possible route of exposure to the test item in humans and is considered suitable to provide the systemic exposure required on this preliminary dose range finding developmental toxicology study.
- Rationale for animal assignment (if not random):
The sperm-positive, assumed pregnant females were allocated to each experimental group (on each mating day) in such a way that the group averages of the body weight were as similar as possible. Females paired with the same male were allocated to different groups on the same mating day.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).
Clinical observations were made at least twice daily at the beginning and end of the working day as practical, or before treatment and after treatment, and once before necropsy.
Pertinent behavioural changes and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable. Evaluated parameters included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
Time schedule for examinations: Body weight of each animal was recorded with precision of 1 g on GD0, 3, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION: Yes
Food was measured with precision of 1 g on GD0, 3, 5, 8, 11, 14, 17 and 20. Average food consumption was calculated for each animal at each interval, including GD5-20 and GD0-20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes. The ovaries and uterus were removed and the pregnancy status ascertained.

OTHER: On GD13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The degree of resorption was described in order to estimate the relative time of death of the conceptus.
Fetal examinations:
- External examinations: Yes: all per litter (live foetuses)
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
The statistical evaluation of all numerical data was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) by an appropriate statistical method, including Bartlett, ANOVA and Duncan, Kruskal-Wallis and Mann-Whitney U tests and Chi2 test, as applicable. The homogeneity of variance between groups was checked by Bartlett`s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was made. If the obtained result was significant Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Significant results with inter-group comparisons were further compared using Kruskal-Wallis and Mann-Whitney U-tests.

Dams were excluded from statistical analysis as follows:
- Sperm positive but non pregnant females (total exclusion) [No. 113 and 102 (100 and 350 mg/kg bw/day, respectively)]
- litter 4506 having one live foetus (exclusion of foetal analysis)

No exclusion was possible in the food consumption data of 113 female, as it was calculated based on cage.

Although these animals were excluded from the statistical analysis the report contains all data from these animals.

The limit for growth retarded foetuses was calculated from the average body weight of the vehicle control foetuses. A foetus was considered as growth retarded if the deviation from the mean control values was greater than minus two fold standard deviation of all control foetuses.

In the present study, this cut-off value was set at 2.61 g.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No mortality occurred during the study.

Clinical signs were observed in all dams at 350 mg/kg bw/day starting from GD8 and consisted of occasionally observed slightly decreased activity (4 of 6), piloerection (4 of 6), hunched back (2 of 6). In addition slightly increased diuresis and minimal yellowish/white discharge from vagina was observed in few occasion in 4 of 6 dams, from GD14.
In one dam (4502) additionally tremor, splayed/unsteady gait, crouching, markedly decreased activity and slightly laboured respiration were observed on GD12 after the treatment.
At 250 mg/kg, slight symptoms were occasionally observed in few animals (e.g. piloerection in 1 of 6 female following the last treatment, slightly increased diuresis in 1 of 6 from GD18, and minimal yellowish/white discharge from vagina in 5 of 6 dams). One of 6 females was symptom-free during the treatment period.
At 100 mg/kg all dams were symptom-free during the treatment period, except minimal yellowish/white discharge from vagina noted for one dam (2506) observed on few occasion from GD11.
In addition, immediately after the treatment minimal salivation and digging of bedding were occasionally observed in all test item treated groups. These observations were regarded to be procedure related.
Occasionally, some small local changes (thin fur) were noticed with low incidence in single female in control group and were regarded as incidental and not related to treatment.

At 350 mg/kg/day, the body weights of dams were lower than control mean (by 14% on GD20) and the differences were statistically significant from GD11-20 (p<0.01). There was a significantly lower maternal body weight gain during the treatment period resulting in lower cumulative value (by 50% for body weight gain and by 51% for corrected gain values). The differences attained statistical significance (p<0.01). The average food consumption during the treatment period was also lower (by about 25% of the control mean) and the differences were statistically significant (p<0.01). The body weight changes are presented in the Text table 4.

At 250 mg/kg/day the body weights were lower than controls (mean by 5% on GD20), and a negative effect on body weight gain was observed during the entire treatment period, resulting in lower cumulative value (by approximately 26% for both body weight gain and corrected gain values). Compared to the control mean, the difference attained statistical significance for body weight gain between GD5-8 and GD5-20 (p<0.01) and for body weight gain between GD11-14 and corrected gain (p<0.05). See Text table 4 below. Compared to the control mean, the food consumption of dams at 250 mg/kg/day was also lower (by about 15%) during the treatment period (p<0.05).

At 100 mg/kg/day the body weights and body weight gains were not statistically different to controls (except gain of GD5-8, p<0.05) but were also slightly lower during the treatment period, resulting in slightly lower cumulative value (by approximately 12% and 8% for body weight gain and corrected gain values, respectively). The food consumption of dams did not differ from the control mean with exception of GD5-8 values (non significant difference).
No test item related observations were recorded at necropsy of dams on GD20 at 350, 250 or 100 mg/kg/day.
Dose descriptor:
dose level:
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There were no toxicological significant differences in intrauterine mortality between test item treated and control females, however at 350 mg/kg bw/day the postimplantation loss was higher than control mean (17.7% versus 4.3%) and was attributed to individual values of one of 6 dams. This dam (4506) had 9 implantation, 6 early and 2 late embryonic loss and only one viable foetus. The differences attained statistical significance for absolute numbers (p<0.05), but not for litter base.

The mean number of corpora lutea and implantation sites was comparable with the controls in all treated groups.

The mean number of viable foetuses was at control level in all treated groups.

The sex distribution of foetuses did not differ between the control and treatment groups, with the exception of 100 and 350 mg/kg/day. At 100 mg/kg/day relatively lower number of male foetuses (36%) and more female foetuses (64%) was found, while at 350 mg/kg/day relatively more male foetuses (62%) and less female foetuses (38%) were counted. The differences were not statistically significant.
There was no foetal death in any of the treated groups.

Pre-implantation loss was similar to control levels in all tested groups.
The weight of foetuses was decreased in all treated groups, by approximately 12-13% (100 mg/kg/day) and 22-23% (250 and 350 mg/kg bw/day), evaluated by both litter mean and dose group mean when compared to the controls. The differences attained statistical significance (p<0.01) in all test item treated groups.

The incidence of body weight retarded foetuses was higher than control in all test item treated groups (by 25%, 64% and 67%, in the Low, Mid and High dose groups, respectively). Compared to the control mean, the difference attained statistical significance (p<0.01) for all dose groups.

The foetuses were subjected only to external examinations.

There was no external abnormality of any of the test item treated or control foetuses.
Dose descriptor:
dose level:
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Under the conditions of this study oral (gavage) administration of N-Butylbenzene-sulfonamide to pregnant Hannover Wistar rats at dose levels of 250 and 350 mg/kg/day, daily from GD5 to 19, produced evident maternal and foetal toxicity. At 100 mg/kg/day mild maternal toxicity and foetal toxicity was observed.
In consultation with the Sponsor, the dose levels recommended for a main developmental toxicity study in the rat are 12, 40 and 120 mg/kg bw/day, at a dose volume of 1 mL/kg.
Executive summary:

The objective of the preliminary study was to assess the effects of the test item N-Butylbenzenesulphonamide on pregnant Hannover Wistar rats and on the developing conceptuses in order to set the dose levels for the main developmental toxicity study.

The dams (one control and 3 treated groups) were treated daily by oral (gavage) administration, from Gestation Day 5 (GD5) up to and including GD19 at dose levels of 100, 250 and 350 mg/kg/day. Control dams were treated with vehicle, polyethylene glycol (PEG 400) only.

Twenty nine sperm positive females were included in the study, six-seven in each test item treated group and nine in the control group.

No mortality occurred during the study. The following clinical signs were noted at 350 mg/kg bw/day starting from GD8: slightly decreased activity (4/6), piloerection (4/6), hunched back (2/6), slightly increased diuresis and minimal yellowish/white discharge from vagina (4/6). In one dam additionally tremor, splayed/unsteady gait, crouching, markedly decreased activity and slightly laboured respiration were observed on GD12 after the treatment. At 250 mg/kg, slight symptoms were occasionally observed in few animals toward the end of the treatment period (e.g. piloerection (1/6), slightly increased diuresis (1/6), and minimal yellowish/white discharge from vagina in (5/6)). At 100 mg/kg, all dams were generally symptom-free during the treatment period. In addition, immediately after the treatment minimal salivation and digging of bedding were occasionally observed in all test item treated groups and were regarded to be procedure related.

Dams at 350 mg/kg/day had lower body weight gain (approximately by 50% both body weight gain between GD5-20 and corrected gain value). The average food consumption was lower than control mean by approximately 25%. At 250 mg/kg/day a negative effect on body weight gain was observed during the entire treatment period, resulting in lower cumulative value (by approximately 26% for both body weight gain between GD5-20 and corrected gain values) and was accompanied by decreased food consumption of dams (by about 15%).

At 100 mg/kg/day the body weights and body weight gains were also slightly lower during the treatment period (by approximately 12% and 8% for body weight gain between GD5-20 and corrected gain values, respectively). The food consumption of dams was close to the control level with exception of GD5-8 values (non significant difference).

No test item related gross pathology findings were recorded at necropsy of dams. No significant differences to control were observed in the intrauterine mortality. At 350 mg/kg/day, postimplantation loss (mostly early intrauterine mortality) was increased, and was attributable to individual values of one female. The mean number of corpora lutea and implantation sites in the test item treated dams did not differ significantly from those of the control means. The mean number of viable foetuses was comparable to control level.

No significant differences were noted in sex distribution of foetuses. The mean weight of foetuses was decreased in all treated groups, by approximately 22-23% (250 and 350 mg/kg bw/day) and 12-13% (100 mg/kg/day), evaluated by both litter mean and dose group mean when compared to the controls. There was no external abnormality of any of the test item treated or control foetuses.

Under the conditions of this study oral (gavage) administration of N-Butylbenzene-sulfonamide to pregnant Hannover Wistar rats at dose levels of 250 and 350 mg/kg/day, daily from GD5 to 19, produced evident maternal and foetal toxicity.

 

At 100 mg/kg/day mild maternal toxicity and foetal toxicity was observed.

 

In consultation with the Sponsor, the dose levels recommended for a main developmental toxicity study in the rat are 12, 40 and 120 mg/kg bw/day, at a dose volume of 1 mL/kg.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
In an OECD Guideline 414 (Prenatal Developmental Toxicity Study) the potential of N-butylbenzenesulphonamide to induce developmental toxicity was determined after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given via diet to time-mated female New Zealand White Rabbits from Days 6 to 29 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 1900, 3800 and 7500 ppm (0, 54, 105, 195 mg/kg bw/day).
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
Test System
Time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalaronne, France). The females arrived on Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating). They were 17-19 weeks old and weighed between 2836 and 4349 g at the initiation of administration.

Animal Identification
At study assignment, each animal was identified by tattoo in the ear.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of administration.

Selection, Assignment, Replacement, and Disposition of Animals
One day before dosing, animals were assigned to groups by a computer-generated random algorithm according to body weights. Each set of females mated on the same date (i.e. 4 sets) was distributed as evenly as possible over the dose groups with body weights within ± 20% of the mean for each set of animals. Animals with a lower than normal relative food intake (based on historical data from rabbits of similar age and of the same strain) were equally distributed across the dose groups, as far as practically feasible. Details of the disposition of all animals were documented in the study records.

Husbandry
Housing
On arrival, females were housed individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm). The cages were equipped with water bottles. The rooms in which the animals were kept was documented in the study records.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group and animal number.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 19-20°C with an actual daily mean relative humidity of 42 to 83%. The values that were outside the targeted range occurred on 9 days in total with a maximum of 83% relative humidity and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
From receipt of animals onwards (i.e post-coitum Day 1), animals received self-pelleted diet without test item. On Days 1, 2 and 3 post-coitum, the self-pelleted diet was supplemented by moistened diet without test item (70 ± 5 grams Standard powder rabbit diet (Global Diet 2030 from Envigo Teklad®, Mucedola, Milanese, Italy) moistened with 50 ± 5 mL tap water, provided in a separate tray/container). The moistened diet was aimed to promote rabbits to eat the dry self-pelleted diet and was refreshed daily. At commencement of treatment, animals had free access to prepared, dry, self-pelleted control or test diet except during designated procedures. In addition, pressed hay (Tecnilab-BMI B.V., Someren, The Netherlands) was provided during the study period.
The feed (without the test item) was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap water was freely available to each animal via water bottles/containers.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It was considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment, animals were provided with shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands).

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.





Route of administration:
oral: feed
Vehicle:
other: diet
Details on exposure:
The test item and control diet were administered to the appropriate animals by inclusion in the diet ad libitum from Day 6 to Day 29 post-coitum, inclusive.
The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test item intake was estimated based on the body weight and food consumption values.
The same diets remained in the food hopper for a maximum of three weeks. Prepared pellets were sieved prior to use to remove grit and prevent spillage
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical Method
Analyses were performed by using a validated analytical procedure (Test Facility Study No. 20175110).

Concentration Analysis
Duplicate sets of samples (approximately 5 g accurately weighed) were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.

Homogeneity Analysis
Duplicate sets of samples (approximately 5 g accurately weighed) were used for homogeneity analysis, the remaining samples were retained at the Test Facility as backup samples. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%. After acceptance of the analytical results, backup samples were discarded.

Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20175110) demonstrated that the test item was stable in the diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20175110.

Diet Analyses
The concentrations analyzed in the pelleted diets of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%).
No test item was detected in the Group 1 diet.
The pelleted diets of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Details on mating procedure:
Time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalaronne, France). The females arrived on Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating). They were 17-19 weeks old and weighed between 2836 and 4349 g at the initiation of administration.
Duration of treatment / exposure:
The test item and control diet were administered to the appropriate animals by inclusion in the diet ad libitum from Day 6 to Day 29 post-coitum, inclusive.
Frequency of treatment:
Daily.
Duration of test:
Animals surviving until scheduled euthanasia were euthanized on Day 29 post-coitum.
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
1 900 ppm
Remarks:
Group 2: 1900 ppm = 54 mg/kg bw/day (Mean over means intake [mg test item/kg body weight])
Dose / conc.:
3 800 ppm
Remarks:
Group 3: 3800 ppm = 105 mg/kg bw/day (Mean over means intake [mg test item/kg body weight])
Dose / conc.:
7 500 ppm
Remarks:
Group 4: 7500 ppm = 195 mg/kg bw/day (Mean over means intake [mg test item/kg body weight])
No. of animals per sex per dose:
Number of females = 22 per group/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
The statement of the testing facility Charles River Laboratories (CRO) regarding dose level rationale for the main OECD 414 study in rabbits with N-butylbenzenesulphonamide (CAS No. 3622-84-2) is available in the attached background material.

- Justification for Test System and Number of Animals:
The New Zealand White rabbit was chosen as the animal model for this study as it is an accepted non-rodent species for developmental toxicity testing by regulatory agencies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
The Study Plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. and approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).
Maternal examinations:
In-life Procedures, Observations, and Measurements – F0-Generation
The in-life procedures, observations, and measurements listed below were performed for parental animals.

Mortality/Moribundity Checks – F0-Generation
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
Animals showing pain, distress or discomfort which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.

Clinical Observations – F0-Generation
Clinical observations were performed at least once daily, beginning on Day 6 post coitum and lasting up to the day prior to necropsy.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
Cage debris was examined to detect abortion or premature birth.

Body Weights – F0-Generation
Animals were individually weighed on Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post coitum.

Food Consumption – F0-Generation
During the acclimatization period, food consumption of dry pellets was measured daily from Day 2 up to Day 5 post-coitum. In addition, food consumption was quantitatively measured for Days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.

Water Consumption – F0-Generation
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

Scheduled Euthanasia – F0-Generation
Animals surviving until scheduled euthanasia were euthanized by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol® 20%) on Day 29 post-coitum. No body weight was recorded at necropsy.

Necropsy – F0-Generation
All animals (including animals sacrificed before planned necropsy) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No tissues, except the uterus, were weighed.
Each ovary and uterine horn of all animals was dissected and examined as quickly as possible to determine:
• The number of corpora lutea.
• The weight of the (gravid) uterus (not for animals sacrificed before planned necropsy).
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
For Female No. 46 that was sacrificed before planned necropsy, these findings are reported in the individual data tables only.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

Ovaries and uterine content:
Each ovary and uterine horn of all animals was dissected and examined as quickly as possible to determine:
• The number of corpora lutea.
• The weight of the (gravid) uterus (not for animals sacrificed before planned necropsy).
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
For Female No. 46 that was sacrificed before planned necropsy, these findings are reported in the individual data tables only.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.
Fetal examinations:
Terminal Procedures – F1-Generation
Method of Euthanasia – F1-Generation
Live fetuses were euthanized by administration of sodium pentobarbital (Euthasol® 20%) into the oral cavity using a small metal feeding tube.

Fetal Examinations (scheduled) – F1-Generation
Litters of females surviving to scheduled necropsy were subjected to detailed external, visceral and skeletal examinations, as described in the following sections.
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External Examinations – F1-Generation
Each viable fetus was examined in detail to detect macroscopic visible abnormalities and their weight was determined (not for fetuses of animals sacrificed before planned necropsy). Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed.
For late resorptions, a gross external examination was performed.

Visceral Examinations – F1-Generation
All fetuses were internally sexed and examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development.
Abnormalities were not collected and fixed in 10% buffered formalin.
The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.
All carcasses, including the carcasses without heads, were eviscerated, skinned, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal Examinations – F1-Generation
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S.
Subsequently, the skeletal examination was done on all fetuses from all groups.
All specimens were archived in glycerin with bronopol as preservative.
A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.



Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and at least 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
Indices:
Reproduction and Developmental Variables
For each group, the following calculations were performed:
Preimplantation loss (%): ([(number of corpora lutea - number of implantation sites)] / [number of corpora lutea]) x 100

Postimplantation loss (%): ([(number of implantation sites - number of live fetuses)] / [number of implantation sites]) x 100

The fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected/litter (%): ([number of viable fetuses affected/litter] / [number of viable festuses/litter]) x 100
Historical control data:
Historical Control Data iare available.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Reduced faeces production was noted across the groups, including controls, however during the first two weeks of treatment the incidence was considered dose-dependently increased for females at 3800 and 7500 ppm. This symptom was considered related to the reduced food consumption of these females. During the third week of treatment, incidence of reduced faeces production at 3800 and 7500 ppm was similar to concurrent controls.
Other clinical signs noted during the treatment period were noted for Female No. 46 (see mortality) or occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item.
One female at 3800 ppm (No. 46) was sacrificed in extremis on Day 23 post coitum. This female lost 7% body weight between Days 18 and 21 post coitum and food consumption was reduced. Red fluid was noted on the manure tray on Days 19-23 post coitum (i.e. Days 14-18 of treatment) and additionally, a hunched posture and severely reduced faeces production were noted for this female from Day 21 post coitum onwards. No macroscopic abnormalities were noted at necropsy. Given the single occurrence in the mid dose group, this mortality was considered to be unrelated to treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 7500 ppm, overall body weight loss was observed on Day 9 post coitum (mean value of - 1% compared to Day 6 post coitum, start of dosing) and although mean body weight gain was observed from Day 12 onwards, mean values remained statistically significantly lower throughout the treatment period when compared with concurrent controls.

At 3800 ppm, mean body weight gain was statistically significantly lower on Days 9-21 post coitum. Mean body weight gain of females at 1900 ppm was also lower than concurrent control, however statistical significance was achieved on Day 12 post coitum only.

Additionally, the mean weight gain corrected for gravid uterus was lower for females at 7500 ppm when compared with concurrent controls (absolute: 260.1 vs 116.1 gram and relative: -7.2% vs -3.2%). No statistical significance was achieved and values generally remained within the historical control range .

No toxicologically relevant changes were observed in mean body weight and uterus weight of treated females up to and including 7500 ppm and weight gain corrected for gravid uterus of treated females up to and including 3800 ppm.

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Based on food consumption during the acclimatization period, all animals with a low food consumption were distributed evenly across the dose groups at study assignment. For most of these females, food consumption recovered to similar values as observed for other animals of the same dose group throughout the study. It was therefore considered that the outcome of the study was not affected by animals with low food consumption at the end of the acclimatization period.
During the treatment period, absolute and relative food consumption of females at 7500 ppm was statistically significantly lower than concurrent controls over Days 6-15 post coitum (up to -47%). In addition, mean food consumption (both absolute and relative) of females at 1900 and 3800 ppm was statistically significantly lower over Days 9-12 post coitum (up to -24% and - 30%, respectively). These effects on food consumption were considered test item-related. At the end of the treatment period, food consumption of females at all dose levels was similar to concurrent controls.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes were observed in mean body weight and uterus weight of treated females up to and including 7500 ppm and weight gain corrected for gravid uterus of treated females up to and including 3800 ppm.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites, and pre- and post implantation loss in the control and test groups were similar and in the range of normal biological variation.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites, and pre- and post implantation loss in the control and test groups were similar and in the range of normal biological variation.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites, and pre- and post implantation loss in the control and test groups were similar and in the range of normal biological variation.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites, and pre- and post implantation loss in the control and test groups were similar and in the range of normal biological variation.
Dead fetuses:
no effects observed
Description (incidence and severity):
Except for one female in the control, 1900, 3800 and 7500 ppm groups each, all females were pregnant with viable litters. Based on the occurrence in the control group and lack of a dose response, these cases of non-pregnancy were considered unrelated to treatment with the test item.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites, and pre- and post implantation loss in the control and test groups were similar and in the range of normal biological variation.
Other effects:
no effects observed
Details on maternal toxic effects:
Based on the results in this prenatal developmental toxicity study, no adverse effects were observed for developmental, maternal toxicity and teratogenicity.
Based on the results, a maternal and developmental No Observed Adverse Effect Level (NOAEL) for N butylbenzenesulphonamide of 7500 ppm (highest dose tested; corresponding to an actual test item intake of 195 mg/kg/day) was established.
Key result
Dose descriptor:
NOAEL
Effect level:
195 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
195 mg/kg bw/day was the highest applied dose.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean combined (male and female) fetal body weights were 38.2, 37.9, 38.4 and 36.3 gram for the control, 1900, 3800 and 7500 ppm groups, respectively.
Mean male, female and combined (male and female) fetal weights per litter were lower (up to 5.0%) at 7500 ppm when compared with concurrent controls. No statistical significance was achieved. The mean fetal combined (male and female) body weight per litter at 7500 ppm (36.3 g fetus) is at the lower end of the 5th percentile of the historical control data .
There were no toxicologically relevant effects on fetal body weights (both sexes) noted after treatment up to 3800 ppm.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no test item-related effects on litter size up to 7500 ppm.
Mean litter sizes were 9.3, 9.7, 8.9 and 9.0 fetuses/litter for the control, 1900, 3800 and 7500 ppm groups, respectively.
The litter incidence of dead fetuses and early or late resorptions was considered unaffected by treatment up to 7500 ppm.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was considered unaffected by treatment up to 7500 ppm.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no test item-related effects on litter size up to 7500 ppm.
Mean litter sizes were 9.3, 9.7, 8.9 and 9.0 fetuses/litter for the control, 1900, 3800 and 7500 ppm groups, respectively.
The litter incidence of dead fetuses and early or late resorptions was considered unaffected by treatment up to 7500 ppm.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no test item-related effects on external morphology following treatment up to 7500 ppm.
External malformations were observed at 1900 and 7500 ppm, but not in the control or 3800 ppm groups.
At 7500 ppm, three fetuses had one or more external malformations. Fetus A073-02 had a cleft lip and palate with underlying skeletal abnormalities. The other malformations occurred in the two fetuses that were dead at scheduled necropsy. Fetus A076-08 had a carpal flexure without apparent skeletal origin and A088-01 had gastroschisis, polymelia, two genital tubercles and no anus. These malformations at 7500 ppm were considered not test item-related as all occurred singly and/or were seen previously in historical control fetuses. Moreover, Fetus A088-01 was also seriously affected viscerally and skeletally and therefore considered an exception of nature.
The four affected fetuses at 1900 ppm (A023-05 and -06 and A028-01 and -09) all had a distended abdomen. At visceral examination this appeared to be caused by cardiac defects (A023-05 and -06) or an enlarged stomach (A028-01 and -09). The exclusive occurrence of distended abdomen in the low dose does not suggest a test item relationship and therefore it was considered to have occurred by chance.
External variations were not observed in this study up to 7500 ppm.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment up to 7500 ppm.
Skeletal malformations occurred in 3 (3), 2 (2), 3 (2), 7 (6) fetuses (litters) in the control, 1900, 3800 and 7500 ppm groups, respectively.
A vertebral anomaly with or without associated rib anomaly was observed most frequently and occurred in 3 (3), 1 (1), 3 (2) and 4 (4) fetuses (litters) in the control, 1900, 3800 and 7500 ppm groups, respectively. As the group distribution of this malformation does not suggest a treatment relationship and this malformation was seen previously in historical control fetuses, it was considered to be spontaneous or origin.
Other malformations observed at 7500 ppm were costal cartilage anomaly, rib anomaly and fused skull bones. These abnormalities occurred singly and were observed in Fetus A071-15, A082-09 and A086-02, respectively. At 1900 ppm, a costal cartilage and a rib anomaly also occurred singly (Fetus A025-04 and A036-04, respectively). Therefore, and as these malformations were seen previously in historical control fetuses, these were considered not related to treatment with the test item.
No other skeletal abnormalities were observed in this study.

Skeletal variations occurred at an incidence of 73.0%, 65.5%, 74.1% and 81.6% per litter in the control, 1900, 3800 and 7500 ppm groups, respectively.
Of the variations, unossified tarsals occurred more frequently at 7500 ppm than at lower dose levels. Incidences were 1.5%, 0.4%, 0.4% and 4.1% per litter in control 1900, 3800 and 7500 ppm groups, respectively. Although this increase was not statistically significant, the high dose value was above the historical control maximum value of 2.1% per litter; it should be recognized that the incidence in the control group (1.5%) was at the high end of the historical control data (mean 0.8%; P95 1.7%). Seven out of eight fetuses with unossified tarsals at the high dose had low body weights ranging from 17.7 to 27.5 gram. These individual fetal weights were generally lower than their litter mean (e.g. Fetus A069-04 weighed 23.3 gram; mean fetal weight of Litter No. 69 was 26.7 gram) and group mean. Consequently, it was considered that this sign of delayed ossification in these fetuses was probably the result of the lower fetal weight and not a direct toxicological effect of the test item.
All other skeletal variations that occurred were considered not test item-related as they occurred infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data.

Visceral malformations:
no effects observed
Description (incidence and severity):
There were no test item-related effects on visceral morphology following treatment up to 7500 ppm.
Visceral malformations occurred in 10 (7), 8 (5), 0 (0) and 4 (3) fetuses (litters) in the control, 1900, 3800 and 7500 ppm groups, respectively.
Other effects:
no effects observed
Description (incidence and severity):
No test item-related changes were noted up to 7500 ppm in any of the remaining maternal parameters investigated in this study (i.e. macroscopic examination, (gravid) uterine weight, uterine contents, number of corpora lutea and implantation sites and pre- and postimplantation loss).
Details on embryotoxic / teratogenic effects:
Based on the results in this prenatal developmental toxicity study, no adverse effects were observed for developmental, maternal toxicity and teratogenicity.
Based on the results, a maternal and developmental No Observed Adverse Effect Level (NOAEL) for N butylbenzenesulphonamide of 7500 ppm (highest dose tested; corresponding to an actual test item intake of 195 mg/kg/day) was established.
Key result
Dose descriptor:
NOAEL
Effect level:
195 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
195 mg/kg bw/day was the highest applied dose.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Based on the results in this prenatal developmental toxicity study, no adverse effects were observed for developmental, maternal toxicity and teratogenicity.

Based on the results, a maternal and developmental No Observed Adverse Effect Level (NOAEL) for N butylbenzenesulphonamide of 7500 ppm (highest dose tested; corresponding to an actual test item intake of 195 mg/kg/day) was established.

FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY

FEMALES (F0-GENERATION)

 

 GROUP 1

CONTROL

GROUP 2

1900 PPM

 GROUP 3

3800 PPM

GROUP 4

7500 PPM

POST COITUM

 DAYS 6-9

MEAN

ST.DEV.

N

147

41.7

21

136

26.8

21

121

35.5

21

78**

33.1

21

 DAYS 9-12

MEAN

ST.DEV.

N

136

37.2

21 

104**

30.3

20

95**

32.7

21 

101**

25.0

21 

 DAYS 12-15

MEAN

ST.DEV.

N

96

41.9

21 

71

30.3

21

70

40.1

21 

64*

30.5

21

 DAYS 15-18

MEAN

ST.DEV.

N

111

43.7

21 

98

34.7

21 

93

40.0

21

91

39.9

21

 DAYS 18-21

MEAN

ST.DEV.

N

120

35.0

21 

120

29.8

21 

114

34.5

21

112

33.1

21 

 DAYS 21-24

MEAN

ST.DEV.

N

97

36.3

21

104

25.6

21 

106

39.7

21

104

28.2

21 

 DAYS 24-27

MEAN

ST.DEV.

N

93

31.9

21 

97

27.0

21

97

26.2

20

99

21.6

21

 DAYS 27-29

MEAN

ST.DEV.

N

103

33.2

21

107

34.3

21 

104

23.1

20

108

25.2

21

 MEAN OF MEANS

113

105

100

95

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

RELATIVE FOOD CONSUMPTION (G/KG BODY WEIGHT/DAY) SUMMARY

FEMALES (F0-GENERATION)

 GROUP 1

CONTROL

GROUP 2

1900 PPM

 GROUP 3

3800 PPM

GROUP 4

7500 PPM

POST COITUM

 DAYS 6-9

MEAN

ST.DEV.

N

40

10.7

21

38

6.8

21

34

9.8

21

22**

9.2

21

 DAYS 9-12

MEAN

ST.DEV.

N

37

9.6

21 

29**

7.3

20

27**

9.3

21 

28**

6.7

21 

 DAYS 12-15

MEAN

ST.DEV.

N

25

10.7

21 

19

8.1

21

20

11.2

21 

18*

8.6

21

 DAYS 15-18

MEAN

ST.DEV.

N

29

11.1

21 

27

9.9

21 

26

10.8

21

25

10.5

21

 DAYS 18-21

MEAN

ST.DEV.

N

32

9.0

21 

33

8.2

21 

31

9.4

21

31

8.8

21 

 DAYS 21-24

MEAN

ST.DEV.

N

25

9.0

21

28

7.5

21 

29

10.2

21

28

7.5

21 

 DAYS 24-27

MEAN

ST.DEV.

N

24

9.7

21 

26

7.8

21

26

6.5

20

26

5.3

21

 DAYS 27-29

MEAN

ST.DEV.

N

26

8.1

21

28

9.3

21 

27

5.4

20

29

6.5

21

 MEAN OF MEANS

30

28

28

26

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

TEST ITEM INTAKE (MG SUBSTANCE/KG BODY WEIGHT/DAY) SUMMARY

FEMALES (F0-GENERATION)

 GROUP 1

CONTROL

GROUP 2

1900 PPM

 GROUP 3

3800 PPM

GROUP 4

7500 PPM

POST COITUM

 DAYS 6-9

MEAN

ST.DEV.

N

0

0.0

21

72

13.0

21

130

37.1

21

167

69.0

21

 DAYS 9-12

MEAN

ST.DEV.

N

0

0.0

21 

55

13.8

20

102

35.3

21 

213

49.9

21 

 DAYS 12-15

MEAN

ST.DEV.

N

0

0.0

21 

37

15.4

21

74

42.4

21 

134

64.6

21

 DAYS 15-18

MEAN

ST.DEV.

N

0

0.0

21 

51

18.8

21 

98

41.2

21

188

78.7

21

 DAYS 18-21

MEAN

ST.DEV.

N

0

0.0

21 

62

15.5

21 

119

35.7

21

230

66.3

21 

 DAYS 21-24

MEAN

ST.DEV.

N

0

0.0

21

53

14.2

21 

112

38.8

21

211

56.2

21 

 DAYS 24-27

MEAN

ST.DEV.

N

0

0.0

21 

49

14.8

21

98

24.5

20

199

39.4

21

 DAYS 27-29

MEAN

ST.DEV.

N

0

0.0

21

53

17.7

21 

104

20.5

20

215

48.4

21

 MEAN OF MEANS

0

54

105

195

BODY WEIGHTS (GRAM) SUMMARY

FEMALES (F0-GENERATION

    

 GROUP 1

CONTROL

GROUP 2

1900 PPM

 GROUP 3

3800 PPM

GROUP 4

7500 PPM

POST COITUM

 DAY 6

 MEAN

ST.DEV.

N

3521

299.4

21

3529

280.9

21

3521

352.8

21

3535

247.8

21

 DAY 9

MEAN

ST.DEV.

N

3613

298.4

21 

3575

291.5

21

3564

327.9

21 

3513

249.4

21 

 DAY 12

MEAN

ST.DEV.

N

3662

286.6

21 

3580

293.3

21

3561

305.9

21 

3550

234.5

21

 DAY 15

MEAN

ST.DEV.

N

3724

284.6

21 

3650

271.0

21 

3588

301.1

21

3581

221.9

21

 DAY 18

MEAN

ST.DEV.

N

3744

290.3

21 

3667

256.5

21 

3628

296.5

21

3625

236.8

21 

 DAY 21

MEAN

ST.DEV.

N

3782

264.2

21

3694

245.9

21 

3667

294.7

21

3655

241.1

21 

 DAY 24

MEAN

ST.DEV.

N

3823

259.7

21 

3744

244.7

21

3707

288.1

20

3691

242.8

21

 DAY 27

MEAN

ST.DEV.

N

3856

267.2

21

3798

225.6

21 

3761

296.1

20

3734

235.0

21

 DAY 29

MEAN

ST.DEV.

N

3902

288.1

21

3839

220.6

21

3801

312.3

20

3756

239.6

21

SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY

 GROUP: 0 PPM   1900 PPM 3800 PPM   7500 PPM

FEMALE FETAL WEIGHTS (g)  

MEAN

ST.DEV.

N

38.1

6.02

21

38.4

3.79

20

38.1

4.63

20

 

37.0

4.63

20

COMBINED FETAL WEIGHTS (g) 

MEAN

ST.DEV.

N

38.2

4.80

21

37.9

4.37

21

38.4

4.32

20

36.3

4.65

21

MACROSCOPIC FINDINGS SUMMARY

FEMALES

 

 GROUP 1

CONTROL

GROUP 2

1900 PPM

GROUP 3

3800 PPM

GROUP 4

7500 PPM

POST COITUM

Animals examined

Animals without findings

22

20 

22

21

22

19

22

17

Animals affected

 2

 1

 3

 5

Lungs

Focus/foci

 

0

 

0

 

1

Liver

Enlarged

0

0

0

1

Gallbladder

Reduced in size

0

0

1

Oviducts

Cyst(s)

0

1

0

0

Spleen

Ectopic splenic tissue

 

0

 

0

 

2

Thymus

Focus/foci

 

1

 

0

 

0

 

0

Skin

Alopecia

 

1

 

0

 

2

 

0

Bone

Front paw missing toe

 

0

 

0

 

0

 

1

SUMMARY OF MATERNAL SURVIVAL AND PREGNANCY STATUS

 Dose group

    1

    2

    3

    4

 

 No.

 %

 No.

 %

 No.

 %

 No.

 %

 FEMALES ON STUDY

 22

 

 22

 

 22

 

 22

 

FEMALES THAT ABORTED

OR DELIVERED

 0

 0.0

 0

 0.0

 0

 0.0

 0

 0.0

FEMALES THAT ABORTED

OR DELIVERED

 0

 0.0

 0

 0.0

 0

 0.0

 0

 0.0

 FEMALES THAT ABORTED

NONGRAVID

GRAVID

0

0

0

0.0

0.0

0.0 

0

0

0.0

0.0

0.0 

0

0

0.0

0.0

0.0 

0

0

0.0

0.0

0.0 

 FEMALES THAT WERE EUTHANIZED

NONGRAVID

GRAVID

0

0

0

0.0

0.0

0.0 

0

0

0.0

0.0

0.0 

1

0

4.5

0

100.0 

0

0

0.0

0.0

0.0 

FEMALES EXAMINED AT

SCHEDULED NECROPSY

NONGRAVID

GRAVID

WITH RESORPTIONS ONLY

WITH VIABLE FETUSES

22

1

21

0

21

100.0

4.5

95.5

0.0

100.0

22

1

21

0

21 

100.0

4.5

95.5

0.0

100.0 

21

1

20

0

20 

95.5

4.8

95.2

0

100.0

22

21

0

21

100.0

4.5

95.5

0

100.0

 TOTAL FEMALES GRAVID

21 

 95.5

 21

 95.5

 21

 95.5

 21

 95.5

SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY

 

 

Sex

 M

 Sex

F

 Viable

fetuses

 Dead

fetuses

Resorptions

Early 

Resorptions

 Late

 Post

implantation

loss

Implantation

sites 

Corpora

lutea 

Pre

implantation

loss 

 Fetal

weights

in grams

No. of

gravid

females 

 1

 Total

Mean

S.D.

104

5.0

2.22 

91

4.3

 2.92

195

9.3

 2.85

0

0.0

0.00 

11

0.5

 0.81

1

0.0

 0.22

12

0.6

0.81 

207

9.9

 2.43

226

10.8

 2.17

19

0.9

 0.94

NA

38.2

 4.80

 21

 2

 Total

Mean

S.D.

108

5.1

2.06

96

4.6

1.72 

204

9.7

 2.10

0

0.0

0.00 

5

0.2

 0.54

2

0.1

0.44 

7

0.3

0.66 

211

10.0

 1.83

228

10.9

 1.35

17

0.8

 1.17

NA

37.9

 4.37

21

 

 3

 Total

Mean

S.D.

83

4.2

1.90 

95

4.8

 1.48

178

8.9

2.00

0

0.0

0.00 

4

0.2

 0.41

8

0.4

0.88 

12

0.6

 0.99

190

0.5 

1.93

198

9.9

1.86 

8

0.4

 0.60

NA

38.4

 4.32

20 
 4

 Total

Mean

S.D.

109

5.2

 1.99

81

3.9

 1.98

190

9.0

 2.60

2

0.1

 0.30

12

0.6

 1.08

3

0.1

 0.48

17

0.8

 1.17

207

9.9

2.29 

224

10.7

1.71 

17

0.8

1.50 

NA

36.3

 4.65

21

 

None significantly different from control group

NA = NOT APPLICABLE

MEAN NUMBER OF VIABLE FETUSES, MEAN NUMBER OF IMPLANTATION SITES, MEAN NUMBER OF CORPORA LUTEA,

FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

-----------------------------------------------------------------------------------------------------------------------------------

1- 0 PPM; 2- 1900 PPM; 3- 3800 PPM; 4- 7500 PPM

SUMMARY OF FETUSES AND LITTERS WITH MALFORMATIONS [ABSOLUTE NO.]

            Fetuses           Litters
 Dose group  1  2  3  4  1  2  3  4

 NUMBER EXAMINED EXTERNALLY

ABDOMEN- DISTENDED

CLEFT LIP

CLEFT PALATE

195

0

0

204

4

0

178

0

0

190

0

1

 21

0

0

0

21

2

0

 20

0

0

0

 21

0

1

1

NUMBER EXAMINED VISCERALLY

KIDNEY(S)- MALPOSITIONED

TESTIS- MALPOSITIONED

ATRIUM- LARGE

ATRIOVENTRICULAR VALVE- ABSENT

KIDNEY(S)- SMALL

HYDROCEPHALY- INTERNAL

DIAPHRAGM- CYST

AORTIC ARCH- NARROW

ATRIAL SEPTUM- DEFECT

LUNG- CYST

195

6

1

1

0

0

0

0

0

1

204

1

4

3

2

1

0

1

1

1

178

0

0

0

0

0

0

0

0

0

190

0

1

0

0

0

1

1

0

0

21

2

3

1

1

0

0

0

0

0

21

1

2

1

1

1

0

1

1

1

20

0

0

0

0

0

0

0

0

0

21

0

1

0

0

0

1

1

0

0

NUMBER EXAMINED SKELETALLY

VERTEBRAL ANOMALY WITH OR WITHOUT

ASSOCIATED RIB ANOMALY

COSTAL CARTILAGE ANOMALY

SKULL BONES- FUSED

RIB ANOMALY

195

3

0

0

0

 204

1

1

0

1

178

3

0

0

190

4

1

1

 21

3

0

0

0

21

1

1

0

 20

2

0

0

0

 21

4

1

1

1

TOTAL NUMBER WITH MALFORMATIONS

EXTERNAL :

SOFT TISSUE :

SKELETAL :

COMBINED :

0

10

12

 

4

8

2

12

 

0

0

3

3

 

1

3

7

10

 

0

7

3

8

 

2

5

2

8

 

0

0

2

2

 

1

2

6

7

SUMMARY OF LITTER PROPORTIONS OF MALFORMATIONS [% PER LITTER]

    DOSE GROUP:  1  2  3  3
    NUMBER OF LITTERS EXAMINED EXTERNALLY 21   21  20  21
 ABDOMEN- DISTENDED

 Mean

S.D.

0

0.0 

1.7

5.25 

0

0.0 

0

0.0 

 CLEFT LIP

 Mean

S.D.

0

0.0 

 0

0.0

0

0.0 

0.8

3.64 

 CLEFT PALATE

Mean

S.D. 

0

0.0 

0

0.0 

0

0.0 

0.8

3.64 

----------------------------------------------------------------------------------------------------------------------------------

1- 0 PPM 2- 1900 PPM 3- 3800 PPM 4- 7500 PPM

None significantly different from control group

SUMMARY OF LITTER PROPORTIONS OF MALFORMATIONS [% PER LITTER]

DOSE GROUP:  1  2  3  3
NUMBER OF LITTERS EXAMINED VISCERALLY 21   21  20  21
 KIDNEY(S)- MALPOSITIONED

 Mean

S.D.

0.9

2.77 

0.4

1.98 

0

0.0 

0

0.0 

 TESTIS- MALPOSITIONED

 Mean

S.D.

2.4

7.55

 1.7

6.10

0

0.0 

0.8

3.64 

 ATRIUM- LARGE

Mean

S.D. 

0.4

1.98 

0.9

3.97 

0

0.0 

0

0.00 

 ATRIOVENTRICULAR VALVE- ABSENT

Mean

S.D. 

0.4

1.98 

 0.9

3.97

 0

0.0

 0

0.0

 KIDNEY(S)- SMALL

Mean

S.D. 

 0

0.0

0.4

1.98 

0

0.0 

 0.0.0
 HYDROCEPHALY- INTERNAL

Mean

S.D. 

 0

0.0

 0

0.0

 0

0.0

0.8

3.64 

 DIAPHRAGM- CYST

 Mean

S.D.

 0

0.0

0.5

2.18 

 0

0.0

 0.5

2.18

 AORTIC ARCH- NARROW

Mean

S.D. 

 0

0.0

0.5

2.42 

0

0.0 

 0

0.0

 ATRIAL SEPTUM- DEFECT

Mean

S.D. 

 0

0.0

 0.5

2.42

 0

0.0

0

0.0 

 LUNG- CYST

mean

S.D. 

 1.6

7.27

0

0.0 

0

0.0 

0

0.0 

-----------------------------------------------------------------------------------------------------------------------------------

1- 0 PPM; 2- 1900 PPM; 3- 3800 PPM; 4- 7500 PPM

None significantly different from control group

SUMMARY OF LITTER PROPORTIONS OF MALFORMATIONS [% PER LITTER]

 DOSE GROUP:  1  2  3  3
   NUMBER OF LITTERS EXAMINED SKELETALLY 21   21  20  21

 VERTEBRAL ANOMALY WITH OR WITHOUT

ASSOCIATED RIB ANOMALY

 Mean

S.D.

1.5

3.72

0.5

2.42

1.5

4.54

2.1

4.65

 COSTAL CARTILAGE ANOMALY

 Mean

S.D.

0

0.0 

 0.5

2.42

0

0.0 

0.3

1.45 

 SKULL BONES- FUSED

Mean

S.D. 

0

0.0 

0

0.0 

0

0.0 

0.6

2.73

 RIB ANOMALY

 Mean

S.D.

0

0.0 

1.0

4.36 

0

0.0 

0.4

1.98 

-----------------------------------------------------------------------------------------------------------------------------------

1- 0 PPM; 2- 1900 PPM; 3- 3800 PPM; 4- 7500 PPM

None significantly different from control group

SUMMARY OF FETUSES AND LITTERS WITH VARIATIONS [ABSOLUTE NO.]

            F E T U S E S        L I T T E R S    
 DOSE GROUP:  2  3  4  1  2  3  4

NUMBER EXAMINED EXTERNALLY

NUMBER WITH FINDINGS

195

0

204

178

 190

0

21

21

20

21

 NUMBER EXAMINED VISCERALLY

RETROCAVAL URETER

RIGHT SUBCLAVIAN- RETROESOPHAGEAL

GALLBLADDER- BILOBED

AORTIC ARCH- SUPERNUMERARY ARTERY

LIVER- CYST(S)

LEFT CAROTID- ORIGINATING FROM BRACHIOCEPHALIC TRUNK

OVARY- CYST(S)

LUNG- ABSENT ACCESSORY LOBE

VISCERA- ATTACHMENT

GALLBLADDER- ABSENT OR SMALL

SPLEEN- PALE

RENAL PAPILLA(E)- ABSENT AND/OR SMALL

LUNG- DISCOLORED

VISCERA- CYST(S)

LIVER- SMALL SUPERNUMERARY LOBE(S)

EYE HEMORRHAGIC

195

7

2

1

2

0

0

0

2

0

1

0

4

1

0

0

204

5

0

0

1

0

7

2

0

3

1

1

2

0

1

0

178

7

0

1

2

1

7

2

2

0

0

0

0

0

0

0

190

3

0

2

0

0

4

2

0

1

1

0

0

0

0

2

21

4

1

1

2

0

0

0

2

0

1

0

2

1

0

0

21

2

0

0

1

0

6

2

0

3

1

1

2

0

1

0

20

3

0

1

2

1

6

2

1

0

0

0

0

0

0

0

21

3

0

2

0

0

4

2

0

1

1

0

0

0

0

1

0

 NUMBER EXAMINED SKELETALLY

13TH FULL RIB(S)

13TH RUDIMENTARY RIB(S)

PELVIC GIRDLE- CAUDAL SHIFT

STERNEBRA(E) #5 AND/OR #6 UNOSSIFIED

STERNEBRA(E) MALALIGNED

CAUDAL VERTEBRAL ANOMALY

METACARPAL(S) AND/OR METATARSAL(S) UNOSSIFIED

TARSAL(S) UNOSSIFIED

PUBIS- UNOSSIFIED

STERNEBRAE FUSED

7TH CERVICAL OSSIFICATION SITE(S)

HYOID BODY AND/OR ARCH(ES) UNOSSIFIED

VERTEBRAL CENTRA- REDUCED OSSIFICATION

SKULL- SUPERNUMERARY SITE

SKULL BONE(S)- SPLIT

SKULL BONE- UNOSSIFIED LINE

7TH CERVICAL FULL RIB(S)

RIB(S)- NODULATED

REDUCED OSSIFICATION OF THE SKULL

STERNEBRA(E)- BRANCHED

PELVIC GIRDLE- CRANIAL SHIFT

195

85

18

27

48

16

1

8

4

2

2

2

3

0

1

0

1

1

0

1

0

204

88

15

33

47

11

0

5

1

1

3

4

1

2

1

0

1

2

0

0

0

178

89

18

22

43

4

0

6

1

0

1

2

1

1

0

0

0

0

1

0

0

0

190

111

10

50

51

12

0

9

8

2

6

0

0

1

2

1

0

0

1

2

1

0

21

19

10

13

14

10

1

4

2

2

2

1

1

0

1

0

1

1

0

1

0

 21

19

10

13

17

10

0

3

1

1

2

2

1

2

1

0

1

1

0

0

0

1

20

19

11

8

15

3

0

4

1

0

1

1

1

1

0

0

0

0

1

0

0

21

21

8

15

13

8

0

4

4

2

4

0

0

1

2

1

0

0

1

1

1

Conclusions:
Based on the results in this prenatal developmental toxicity study, no adverse effects were observed for developmental, maternal toxicity and teratogenicity.
Based on the results, a maternal and developmental No Observed Adverse Effect Level (NOAEL) for N butylbenzenesulphonamide of 7500 ppm (highest dose tested; corresponding to an actual test item intake of 195 mg/kg/day) was established.
Executive summary:

In an OECD Guideline 414 (Prenatal Developmental Toxicity Study) the potential of N-butylbenzenesulphonamide to induce developmental toxicity was determined after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given via diet to time-mated female New Zealand White Rabbits from Days 6 to 29 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 1900, 3800 and 7500 ppm (0, 54, 105,195 mg/kg bw/day).

Based on the results in this prenatal developmental toxicity study, no adverse effects were observed for developmental, maternal toxicity and teratogenicity.

Based on the results, a maternal and developmental No Observed Adverse Effect Level (NOAEL) for N butylbenzenesulphonamide of 7500 ppm (highest dose tested; corresponding to an actual test item intake of 195 mg/kg/day) was established.

Time-mated female New Zealand White rabbits were treated with N-butylbenzenesulphonamide from Day 6 to 29 post-coitum, inclusive by dietary administration at dose levels of 1900, 3800 and 7500 ppm (0, 54, 105,195 mg/kg bw/day). The rabbits of the control group received the prepared pelleted rabbit diet without test item. Analysis of diet preparations confirmed that the achieved dietary concentrations of N-butylbenzenesulphonamide were in agreement with the target concentrations and that the test item was homogeneously distributed in the diet.

Maternal findings

No mortality occurred during the study period that was considered to be related to treatment with the test item.

During the treatment period, absolute and relative food consumption of females at 7500 ppm was statistically significantly lower than concurrent controls over Days 6-15 post coitum (up to -47%). In addition, mean food consumption (both absolute and relative) of females at 1900 and 3800 ppm was statistically significantly lower over Days 9-12 post coitum (up to -24% and - 30%, respectively). In subsequent intervals, food consumption recovered to levels similar to concurrent controls by the end of the treatment period.

Overall body weight loss was observed at 7500 ppm on Day 9 post coitum (mean value of -1% compared to Day 6 post coitum, start of dosing) and although mean body weight gain was observed from Day 12 onwards, mean values remained statistically significantly lower throughout the treatment period when compared with concurrent controls. At 3800 ppm, mean body weight gain was statistically significantly lower on Days 9-21 post coitum. Mean body weight gain of females at 1900 ppm was also lower than concurrent control, however statistical significance was achieved on Day 12 post coitum only.

As food consumption of treated females recovered to levels similar to concurrent controls by the end of the treatment period and the effects on body weight gain did not result in statistically significant changes in body weight or weight gain corrected for gravid uterus, these changes in food intake and body weight gain at the high dose were considered to be non-adverse up to and including 7500 ppm.

Clinical signs at 3800 and 7500 ppm were reduced faeces production for females during the first two weeks of treatment, which was considered related to the reduced food consumption during this period. As the incidence of this symptom was similar to the incidence in the concurrent control group during the last week of treatment, this observation was considered to be non-adverse.

No test item-related changes were noted up to 7500 ppm in any of the remaining maternal parameters investigated in this study (i.e. macroscopic examination, (gravid) uterine weight, uterine contents, number of corpora lutea and implantation sites and pre- and postimplantation

loss).

Note: Food consumption was markedly reduced during the first three days of treatment which resulted in a mean body weight loss of 1%. For individual animals, a body weight loss up to 5% was noted on Day 9 post coitum. This palatability effect was considered to be a doselimiting factor, as higher concentrations could induce even more body weight loss if the food intake would be further reduced and this could result in early euthanization of animals for welfare reasons. In the dose range finder study similar effects were also noted at 5000 ppm, however at this concentration values still recovered to normal levels by the end of the treatment period. Based on these results, concentrations higher than 7500 ppm were considered inappropriate for a study in pregnant rabbits.

Fetal findings

Mean male, female and combined (male and female) fetal weights were lower (up to 5.0%) at 7500 ppm when compared with concurrent controls. In addition, a higher litter incidence of the variation ‘unossified tarsals’ (4.1% vs 1.5%) was noted at this dose level. Although this increase was not statistically significant, the high dose value was above the historical control maximum value of 2.1% per litter. Based on the individual fetal weights of affected fetuses, this sign of delayed ossification in these fetuses was considered a fetal weight effect and not a direct toxicological effect of the test item. Moreover, as the variation was observed in only 8 out of 190 fetuses and the difference in mean fetal weight did not reach statistical significance, these effects were considered to be non-adverse.

No toxicologically significant changes were noted up to 7500 ppm in any of other the developmental parameters investigated in this study (i.e. the number of live and dead fetuses, early and late resorptions, sex ratio, and external and visceral malformations and developmental variations).

Based on the results in this prenatal developmental toxicity study no adverse effects were observed for developmental, maternal toxicity and teratogenicity.

Based on the results, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for N-butylbenzenesulphonamide was established as being 7500 ppm (highest dose tested; corresponding to an actual test item intake of 195 mg/kg/day).

Endpoint:
developmental toxicity
Remarks:
DRF (Dose Range Finder)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
7 June 2019 to 5 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline followed
Principles of method if other than guideline:
A dose range finder (Test Facility Reference No. 20175108) was conducted to select dose levels for the Prenatal Developmental Toxicity Study (Test Facility Study No. 20175109).
No guidelines were applicable as this study was intended for dose level selection purposes only.
If not mentioned otherwise, test system, procedures and techniques were identical to those used during the main study.
Dosing of the DRF was initiated on 12 Jun 2019. The in-life phase of the DRF was completed on 05 Jul 2019.
GLP compliance:
no
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
Test System
On 07 Jun 2019, time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalaronne, France). The females arrived on Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating). They were 17-19 weeks old and weighed between 3236 and 4065 g at the initiation of administration.

One day before dosing, animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals preferably within ± 20% of the mean, if feasible (the actual maximum weight range applied will be reported). Animals with a lower than normal relative food intake (based on historical data from rabbits of similar age and of the same strain) were equally distributed across the dose groups, as far as practically feasible.
Details were recorded in the raw data. The disposition of all animals was documented in the study records.

From receipt of animals onwards (i.e. post-coitum Day 1), animals received self-pelleted diet without test item supplemented by moistened diet without test item (70 ± 5 grams Standard powder rabbit diet (Global Diet 2030 from Envigo Teklad®, Mucedola, Milanese, Italy) moistened with 50 ± 5 mL tap water, provided in a separate tray/container). The moistened diet was aimed to promote rabbits to eat the dry self-pelleted diet. Once rabbits ate the dry self-pelleted diet to a level to be expected for rabbits of this age and strain, only the dry selfpelleted diet were provided to the respective animals. At commencement of treatment, animals only received dry self-pelleted control or test diet.

The actual daily mean temperature during the study period was 19 to 20°C with an actual daily mean relative humidity of 53 to 94%. The values that were outside the targeted range occurred on 10 days in total with a maximum of 94% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study.

Route of administration:
oral: feed
Vehicle:
other: diet
Details on exposure:
Diet Preparations
Analysis of test item in diet for concentration, homogeneity, and stability were not performed. However, to limit the impact, the diet preparation was performed with approved procedures and documented in detail. Preparations were used within three weeks after preparation when kept at room temperature. Homogeneity and stability of the diet under test conditions was demonstrated in the analytical method development and validation study (Test Facility Study No. 20175110).
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Analysis of test item in diet for concentration, homogeneity, and stability were not performed.
The test item has a purity of 99.9%, dose calculations were not corrected for purity.
Details on mating procedure:
On 07 Jun 2019, time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalaronne, France). The females arrived on Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
The test item and vehicle were administered to the appropriate animals by inclusion in the diet ad libitum from Day 6 to Day 29 post-coitum, inclusive.
Frequency of treatment:
Daily.
Duration of test:
29 days.
Dose / conc.:
0 ppm
Remarks:
Group No. 1: Prepared pelleted rabbit diet without test item.
Dose / conc.:
1 250 ppm
Remarks:
Group No. 2:
Mean over means intake [mg test item/kg body weight] = 37
(mean range indicated within brackets) = (29-48)
Dose / conc.:
2 500 ppm
Remarks:
Group No. 3:
Mean over means intake [mg test item/kg body weight] = 67
(mean range indicated within brackets) = (52-81)
Dose / conc.:
5 000 ppm
Remarks:
Group No. 4:
Mean over means intake [mg test item/kg body weight] = 131
(mean range indicated within brackets) = (86-166)
No. of animals per sex per dose:
6 female rabbits/group.
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected based on the results of a tolerability study with N-butylbenzenesulphonamide in rabbits by oral gavage (Test Facility Study No. 20175107, dose levels of 580, 1740 and 5000 ppm):

• The dose level 580 ppm was well tolerated, as there were no differences between the control and treated group with respect to feed intake, body weight development and clinical signs that were considered to be test item-related.

• At 1740 ppm, one animal had slight body weight loss (1-2%) and for the other two animals a reduced body weight gain was noted when compared with concurrent control (3-4% vs. 8-9% on Day 14 of exposure). Food consumption was similar between control animals and animals treated at 1740 ppm.

• At 5000 ppm, body weight loss (up to 6%) and notably reduced food consumption was noted in 3/3 treated animals during the initial four days of treatment. The body weight loss persisted during treatment, but did not deteriorate further and food consumption appeared to recover to levels similar to controls after approximately one week of treatment.

Based on these results, 5000 ppm was selected as the maximum dose level for the dose range finding study. Based on the observed effects on body weight and food consumption at 5000 ppm, it was not considered necessary to test higher dose levels in the tolerability study.
Maternal examinations:
In-Life Procedures, Observations and Measurements
During the acclimatization period, the dry self-pelleted diet was weighed to measure the food consumption for Days 2-3, 3-4 and 4-5 post coitum to determine whether consumption of the dry self-pelleted diet was at a level to be expected for rabbits of this age and strain.
Fetal examinations:
Terminal Procedures – F1-Generation
Each viable fetus of animals surviving to planned necropsy was externally examined in detail and weighed. All live fetuses were euthanized by administration of sodium pentobarbital (Euthasol® 20%) into the oral cavity using a small metal feeding tube.
Nonviable fetuses of animals surviving to planned necropsy (the degree of autolysis is minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed.
Sex was not determined. No visceral (internal) or skeletal examination was performed. As no malformations were observed, all fetuses and late resorptions were discarded.
Historical control data:
Historical control data are available.
Clinical signs:
no effects observed
Description (incidence and severity):
Reduced faeces production was observed across the groups, but no dose-related trend was noted.
For one female at 1250 ppm (No. 9), red fluid (most likely blood) was noted on the manure tray on Days 27 and 28 post coitum.
Other findings that were recorded occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For detailed information on body weight, food consumption and test item intake see attached document in the IUCLID field ‘attached background material’ (pdf:20175108 body weight, food consumption and test item intake end of in-life).

At 5000 ppm, mean body weight loss was noted for females on Day 9 post coitum vs Day 6 (start dosing), with 4/5 pregnant females showing body weight loss ranging between 1 and 4%. Mean body weight gain showed an upward trend on subsequent measurement intervals, with comparable values to concurrent controls from Day 18 post coitum onwards.

At 2500 ppm, there was on average no body weight gain up to Day 12 post-coitum (2/5 pregnant females showed minimal body weight loss, i.e. 1%), but mean body weight gain showed an upward trend on subsequent measurement intervals.

At 1250 ppm, mean body weight gain was within the normal range.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
For detailed information on body weight, food consumption and test item intake see attached document in the IUCLID field ‘attached background material’ (pdf:20175108 body weight, food consumption and test item intake end of in-life).

At 5000 ppm, mean absolute and relative food consumption was statistically significantly lower (up to 55%) than control means over Days 6-12 post-coitum, but was (slightly) higher than control means on several subsequent measurement intervals (statistically significant over Days 24-27); a clear dose-related trend over the dose groups was absent.

At 2500 ppm, a similar trend in absolute and relative food consumption was recorded as seen at 5000 ppm, with means being statistically significantly different over Days 9-12 only.

At 1250 ppm, relative food intake was also statistically significantly lower over Days 9-12.
Absolute food intake appeared generally higher than control means from Day 12 onwards, but when corrected for body weight (i.e. relative food consumption) these differences were less
apparent.

Mean food intake of control animals was considered normal based on historical control data.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Gravid uterus weight and weight gain corrected for gravid uterus of treated animals remained in the same range as controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
One female each at 1250, 2500 and 5000 ppm was non-gravid (Nos. 7, 17 and 19, respectively). All gravid females had live fetuses. The numbers of pregnant females, corpora lutea and implantation sites in the control and test groups were similar and in the range of normal biological variation.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
At 2500 and 5000 ppm, pre-implantation loss was statistically significantly higher than control mean. At 2500 ppm, this concerned animal Nos. 14, 15 and 18, with pre-implantation loss percentages of 64, 27 and 42% respectively. At 5000 ppm, this concerned Nos. 20, 22, 23 and 24 with pre-implantation loss percentages of 7, 7, 11 and 10%, respectively. As the mean pre-implantation loss incidence at 5000 ppm remained within the historical control range and a dose-related trend was absent, it was considered that this finding did not represent an effect of the test item.
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
All gravid females had live fetuses.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites in the control and test groups were similar and in the range of normal biological variation.
Other effects:
not examined
Dose descriptor:
LOAEC
Effect level:
5 000 ppm
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other:
Remarks on result:
other:
Remarks:
Based on the results of the dose range finder, it was concluded that a dose level of 5000 ppm did induce an effect on body weight and food consumption.
Abnormalities:
effects observed, treatment-related
Localisation:
other:
Description (incidence and severity):
Based on the results of the dose range finder, it was concluded that a dose level of 5000 ppm did induce an effect on body weight and food consumption.
Fetal body weight changes:
not examined
Description (incidence and severity):
Fetal body weights were considered not affected by treatment with the test item.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Litter sizes were within normal limits for all groups.
Changes in sex ratio:
not examined
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter sizes were within normal limits for all groups.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
External examination of the fetuses did not show any abnormalities.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Dose descriptor:
NOAEL
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
not specified
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
5000 ppm = approx. 131 mg test item/kg body weight
Abnormalities:
no effects observed
Developmental effects observed:
no

Based on the results of the dose range finder, it was concluded that a dose level of 5000 ppm did induce an effect on body weight and food consumption. Values recovered to normal levels by the end of treatment and no secondary effects were noted on fetal development, exposure to 5000 ppm was therefore considered not to induce adverse effects on maternal or developmental parameters and was considered insufficient as highest dose in the main study.

However, as it could not be excluded that the effect on food consumption and body weight would increase and induce excessive toxicity at higher ppm levels, concentrations higher than 7500 ppm were considered inappropriate for a study in pregnant rabbits.

Selected dose levels for the main study were 1900, 3800 and 7500 ppm.

Conclusions:
Based on the results of the dose range finder, it was concluded that a dose level of 5000 ppm did induce an effect on body weight and food consumption. Values recovered to normal levels by the end of treatment and no secondary effects were noted on fetal development, exposure to 5000 ppm was therefore considered not to induce adverse effects on maternal or developmental parameters and was considered insufficient as highest dose in the main study.
However, as it could not be excluded that the effect on food consumption and body weight would increase and induce excessive toxicity at higher ppm levels, concentrations higher than 7500 ppm were considered inappropriate for a study in pregnant rabbits.
Selected dose levels for the main study were 1900, 3800 and 7500 ppm.
Executive summary:

A dose range finder (Test Facility Reference No. 20175108) was conducted to select dose levels for the Prenatal Developmental Toxicity Study in rabbits (Test Facility Study No. 20175109). No guidelines were applicable as this study was intended for dose level selection purposes only.

Six female rabbits per group received doses of 1250, 2500 or 5000 ppm (Approx. 37, 67 or 131 mg test item/kg body weight).

Each viable fetus of animals surviving to planned necropsy was externally examined in detail and weighed. Nonviable fetuses of animals surviving to planned necropsy (the degree of autolysis is minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed. Sex was not determined. No visceral (internal) or skeletal examination was performed.

Based on the results of the dose range finder, it was concluded that a dose level of 5000 ppm did induce an effect on body weight and food consumption. Values recovered to normal

levels by the end of treatment and no secondary effects were noted on fetal development, exposure to 5000 ppm was therefore considered not to induce adverse effects on maternal or developmental parameters and was considered insufficient as highest dose in the main study.

However, as it could not be excluded that the effect on food consumption and body weight would increase and induce excessive toxicity at higher ppm levels, concentrations higher than 7500 ppm were considered inappropriate for a study in pregnant rabbits.

Selected dose levels for the main study were 1900, 3800 and 7500 ppm.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
120 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A key developmental toxicity study was performed to assess the effects of the test item N-butylbenzenesulphonamide on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy (Grósz, 2014a). The dams (one control and 3-treated groups) were treated daily by oral (gavage) administration, from gestation day GD6 up to and including GD19. Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20.The dose levels were 12, 40 and 120 mg/kg bw/day. Control dams were treated with vehicle polyethylene glycol (PEG 400) only. The number of confirmed pregnant, evaluated dams in the dose groups treated at 12, 40 and 120 mg/kg bw/day was 24 in each of group and 25 in the control group. There was no unscheduled mortality during the study. There were no toxicologically significant clinical signs noted following administration of test item. Body weight gain of dams at 120 mg/kg bw/day was lower than control mean during the treatment period (by 15 and 30% for absolute and net mean values, respectively) and was accompanied by slightly decreased food consumption. No test item related macroscopic changes were found at necropsy of dams on GD20, except few large placentae at 120 mg/kg bw/day (6 foetal and 2 litter incidences, respectively). Mild to moderate dilatation of chorionic vessels was observed in all examined placentas from the High dose group. There were no test-item related significant differences between the treated and control animals regarding the pre-implantation loss or early embryonic death, post-implantation loss, or total intrauterine mortality. The mean number of viable foetuses was comparable with the control mean. The sex distribution of foetuses did not differ significantly from the controls. The weight of foetuses at 120 mg/kg bw/day was decreased by approximately 9% evaluated by both litter mean and group mean when compared to the controls. The differences attained statistical significance. No significant differences in body weight were observed between male and female foetuses at the same dose level. There were no foetal external, visceral or skeletal abnormalities ascribed to the test item administration. The No Observed Adverse Effect Level (NOAEL) under the conditions of this study for maternal toxicity was found to be 40 mg/kg bw/day and for foetal developmental toxicity 120 mg/kg bw/day.

A supporting dose range finding study was performed in pregnant Hannover Wistar rats from Gestation Day 5 (GD5) up to and including GD19 at dose levels of 100, 250 and 350 mg/kg/day (Grósz, 2014b). Twenty nine sperm positive females were included in the study, six-seven in each test item treated group and nine in the control group. No mortality occurred during the study, however clinical signs were noted at 350 mg/kg bw/day starting from GD8 and at 250 mg/kg more toward the end of the treatment period. Dams at 350 mg/kg/day had lower body weight gain and he average food consumption was lower than control. At 250 mg/kg/day a negative effect on body weight gain was observed during the entire treatment period, resulting in lower cumulative value and was accompanied by decreased food consumption of dams. At 100 mg/kg/day the body weights and body weight gains were also slightly lower during the treatment period; the food consumption of dams was close to the control level with exception of GD5-8 values. No test item related gross pathology findings were recorded at necropsy of dams. No significant differences to control were observed in the intrauterine mortality. At 350 mg/kg/day, postimplantation loss was increased, and was attributable to individual values of one female. The mean number of corpora lutea and implantation sites in the test item treated dams did not differ significantly from those of the control means. The mean number of viable foetuses was comparable to control level. No significant differences were noted in sex distribution of foetuses. The mean weight of foetuses was decreased in all treated groups. There was no external abnormality of any of the test item treated or control foetuses.

A preliminary study was conducted in pregnant rabbits (6/group) dosed via oral gavage from Gestation Day 6 -27 to determine the Maximum Tolerated Dose (MTD) and to assess the effects of the test item on pregnant females and on the developing conceptus at 0, 10, 20 and 60 mg/kg bw in corn oil vehicle. Based on the high number of maternal mortalities in all test item treated groups, this pilot study is considered to be invalid. The data of this study indicate there was an overall effect related to the dosing procedures on the feeding and body weight changes of all groups, which would mask or distort any possible test item effects. Consequently, no further conclusion can be drawn from this study (Disregarded study).

A supporting DRF (dose range finder) (Test Facility Reference No. 20175108) was conducted to select dose levels for the Prenatal Developmental Toxicity Study in rabbits (Test Facility Study No. 20175109). No guidelines were applicable as this study was intended for dose level selection purposes only.

Six female rabbits per group received doses of 1250, 2500 or 5000 ppm (Approx. 37, 67 or 131 mg test item/kg body weight).

Each viable fetus of animals surviving to planned necropsy was externally examined in detail and weighed. Nonviable fetuses of animals surviving to planned necropsy (the degree of autolysis is minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed. Sex was not determined. No visceral (internal) or skeletal examination was performed.

Based on the results of the DRF (dose range finder), it was concluded that a dose level of 5000 ppm did induce an effect on body weight and food consumption. Values recovered to normal levels by the end of treatment and no secondary effects were noted on fetal development, exposure to 5000 ppm was therefore considered not to induce adverse effects on maternal or developmental parameters and was considered insufficient as highest dose in the main study.

However, as it could not be excluded that the effect on food consumption and body weight would increase and induce excessive toxicity at higher ppm levels, concentrations higher than 7500 ppm were considered inappropriate for a study in pregnant rabbits.

Selected dose levels for the main study were 1900, 3800 and 7500 ppm.

In an OECD Guideline 414 (Prenatal Developmental Toxicity Study) the potential of N-butylbenzenesulphonamide to induce developmental toxicity was determined after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given via diet to time-mated female New Zealand White Rabbits from Days 6 to 29 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 1900, 3800 and 7500 ppm (0, 54, 105, 195 mg/kg bw/day).

Based on the results in this prenatal developmental toxicity study, no adverse effects were observed for developmental, maternal toxicity and teratogenicity.

Based on the results, a maternal and developmental No Observed Adverse Effect Level (NOAEL) for N butylbenzenesulphonamide of 7500 ppm (highest dose tested; corresponding to an actual test item intake of 195 mg/kg/day) was established.

 

Time-mated female New Zealand White rabbits were treated with N-butylbenzenesulphonamide from Day 6 to 29 post-coitum, inclusive by dietary administration at dose levels of 1900, 3800 and 7500 ppm (0, 54, 105,195 mg/kg bw/day). The rabbits of the control group received the prepared pelleted rabbit diet without test item. Analysis of diet preparations confirmed that the achieved dietary concentrations of N-butylbenzenesulphonamide were in agreement with the target concentrations and that the test item was homogeneously distributed in the diet.

Maternal findings

No mortality occurred during the study period that was considered to be related to treatment with the test item.

During the treatment period, absolute and relative food consumption of females at 7500 ppm was statistically significantly lower than concurrent controls over Days 6-15 post coitum (up to -47%). In addition, mean food consumption (both absolute and relative) of females at 1900 and 3800 ppm was statistically significantly lower over Days 9-12 post coitum (up to -24% and - 30%, respectively). In subsequent intervals, food consumption recovered to levels similar to concurrent controls by the end of the treatment period.

Overall body weight loss was observed at 7500 ppm on Day 9 post coitum (mean value of -1% compared to Day 6 post coitum, start of dosing) and although mean body weight gain was observed from Day 12 onwards, mean values remained statistically significantly lower throughout the treatment period when compared with concurrent controls. At 3800 ppm, mean body weight gain was statistically significantly lower on Days 9-21 post coitum. Mean body weight gain of females at 1900 ppm was also lower than concurrent control, however statistical significance was achieved on Day 12 post coitum only.

As food consumption of treated females recovered to levels similar to concurrent controls by the end of the treatment period and the effects on body weight gain did not result in statistically significant changes in body weight or weight gain corrected for gravid uterus, these changes in food intake and body weight gain at the high dose were considered to be non-adverse up to and including 7500 ppm.

Clinical signs at 3800 and 7500 ppm were reduced faeces production for females during the first two weeks of treatment, which was considered related to the reduced food consumption during this period. As the incidence of this symptom was similar to the incidence in the concurrent control group during the last week of treatment, this observation was considered to be non-adverse.

No test item-related changes were noted up to 7500 ppm in any of the remaining maternal parameters investigated in this study (i.e. macroscopic examination, (gravid) uterine weight, uterine contents, number of corpora lutea and implantation sites and pre- and postimplantation loss).

Note: Food consumption was markedly reduced during the first three days of treatment which resulted in a mean body weight loss of 1%. For individual animals, a body weight loss up to 5% was noted on Day 9 post coitum. This palatability effect was considered to be a dose limiting factor, as higher concentrations could induce even more body weight loss if the food intake would be further reduced and this could result in early euthanization of animals for welfare reasons. In the dose range finder study similar effects were also noted at 5000 ppm, however at this concentration values still recovered to normal levels by the end of the treatment period. Based on these results, concentrations higher than 7500 ppm were considered inappropriate for a study in pregnant rabbits.

Fetal findings

Mean male, female and combined (male and female) fetal weights were lower (up to 5.0%) at 7500 ppm when compared with concurrent controls. In addition, a higher litter incidence of the variation ‘unossified tarsals’ (4.1% vs 1.5%) was noted at this dose level. Although this increase was not statistically significant, the high dose value was above the historical control maximum value of 2.1% per litter. Based on the individual fetal weights of affected fetuses, this sign of delayed ossification in these fetuses was considered a fetal weight effect and not a direct toxicological effect of the test item. Moreover, as the variation was observed in only 8 out of 190 fetuses and the difference in mean fetal weight did not reach statistical significance, these effects were considered to be non-adverse.

No toxicologically significant changes were noted up to 7500 ppm in any of other the developmental parameters investigated in this study (i.e. the number of live and dead fetuses, early and late resorptions, sex ratio, and external and visceral malformations and developmental variations).

Based on the results in this prenatal developmental toxicity study no adverse effects were observed for developmental, maternal toxicity and teratogenicity.

Based on the results, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for N-butylbenzenesulphonamide was established as being 7500 ppm (highest dose tested; corresponding to an actual test item intake of 195 mg/kg/day).

Justification for classification or non-classification

Based on the results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC) and CLP regulation (EC No. 1272/2008 of 16 December 2008), N-butylbenzenesulphonamide does not have to be classified and has no obligatory labelling requirement for reproductive and developmental toxicity.

Additional information