2-(1,1-dimethylpropyl)phenol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD gudieline 422 study on read across analogue o-sec butyl phenol

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Japanese Ministry of Health Study - English translation - well documented

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

not specified

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc
- Age at study initiation: 8 wks
- Mean Body Weight at study initiation: Males: 300+/- 6 g; Females: 220 +/-7.5 g
- Housing: individually in metallic cages. From day 14 of gestation (the day sperm was confirmed = Day 0 of gestation), dams were housed in breeding cages for rats and paper pulp chips (ALPHA-dri, Kasho Co., Ltd.) supplied ad libitum as bedding.
- Diet (ad libitum): pelleted diet (CE-2, CLEA Japan, Inc.)
- Water (ad libitum): tap water (supplied from Waterworks Department, Hadano-shi)
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1°C
- Humidity (%): 50-65%
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hours a day (from 7:00 to 19:00)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test article was dissolved in corn oil
Amount administered (if gavage): 2ml/kg

Details on mating procedure:

Males and females in the same group were housed together on 1:1 basis from the evening of day 15 of administration for 14 days at maximum
Copulation was confirmed by examination for the presence of vaginal plug and sperms in vaginal smears, and the females for which copulation was confirmed were separated from males and housed individually thereafter.
Sperm in vaginal smear referred to as day 0 of pregnancy
From day 14 of gestation (the day sperm was confirmed = Day 0 of gestation), dams were housed in breeding cages for rats and paper pulp chips
(ALPHA-dri, Kasho Co., Ltd.) supplied ad libitum as bedding.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the test article in the dose formulations were confirmed by Hatano Research Institute. No detailed information.

Duration of treatment / exposure:

Single daily gavage exposures, for 42 days males.
28 days plus gestation period up to day 3 lactation in females

Frequency of treatment:

Once daily

Details on study schedule:

Test article was administered once daily to animals 2 weeks before mating, 2 weeks during the mating period.
Administration was repeated once daily for 2 weeks after the end of the mating period for males and throughout the gestation period up to day 3 of
lactation after delivery for females.

Remarks:

Doses / Concentrations:
0 (vehicle control), 12, 60 and 300 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

n= 13 males, n= 13 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels of this study were selected based on the preliminary test in which o-sec-butylphenol was administered
orally by gavage repeatedly for 14 days at dose levels of 0 (corn oil), 30, 100 or 300 mg/kg to groups of rats of the same strain as this study each
group consisting of 5 males and 5 females. The high dose level in the main study was set at 300 mg/kg, at which effects were observed in general condition, body weight and liver weight in males and females.

Positive control:

None

Parental animals: Observations and examinations:

All males and females were observed at least once a day

BODY WEIGHT: All males and females were weighed once a week during the study period (Days 1, 8, 15, 22, 29, 36 and 42 of administration for
males and days 1, 8 and 15 of administration for females). The females that were housed together with males were weighed also on day 22 of
administration. Copulated females were weighed on days 0, 7, 14 and 20 of gestation and delivered females were weighed on days 0 and 4 of
lactation.

FOOD WEIGHT AND CONSUMPTION: For all males and females, weight of the feed was measured on the same days as for body weight measurement, and the food consumption from the day of measurement to the day of next measurement calculated. During the 2-week mating period, food
consumption was not measured. Food consumption was measured from day 0 to day 7, day 7 to day 14 and day 14 to day 20 of gestation for
copulated females and from day 0 to day 4 of lactation for delivered females.

Oestrous cyclicity (parental animals):

The number of estruses during the mating period were determined.

Sperm parameters (parental animals):

The testes and epididymides were weighed

Litter observations:

On days 0 and 4 of lactation, body weight of pups was measured for each litter by sex (litter weight) and (litter weight / number of pups) was
calculated for each litter.

Postmortem examinations (parental animals):

GROSS PATHOLOGY OF MAJOR ORGANS WAS PERFORMED FOR FEMALES AND MALES
The following organs were weighed and the ratio to body weight (relative organ weight) calculated: brain, heart, thymus, liver, kidneys, spleen,
adrenals, testes and epididymides.

FEMALES: For all pregnant and non-pregnant females, the ovary and uterus were excised, the number of implantations was counted using the
uterus, and the females that showed implantations were regarded as pregnant females. For the ovary, the number of corpora lutea was counted
under a stereoscopic microscope, and the ovary was fixed in Bouin’s solution and preserved. The ovaries of the non-pregnant females were
subjected to histopathological examination. The following organs were weighed and fixed in 10 w/v% formalin and preserved: brain, heart,
thymus, liver, kidneys, spleen and adrenals.

MALES: The testes and epididymides were fixed in Bouin’s solution and preserved while the other organs, urinary bladder, lungs and stomach were
fixed and preserved in 10 w/v% formalin. For the males in the control group and the high dose group, these fixed organs were subjected to
histopathological examination. Other than these organs, histopathological examination was not done since they were judged to have no effects
from administration of the test article.

Postmortem examinations (offspring):

On day 4 of lactation, all pups were euthanized by ether inhalation and necropsied, and external and internal organs observed macroscopically.
The pups that died on delivery were autopsied and the external and internal organs observed macroscopically for the presence or absence of
abnormalities.

Statistics:

The copulation index and fertility index were analyzed by the chi-square test with Yates’ correction.
Histopathological findings: graded data were analyzed by Mann-Whitney’s U test; the sum of the positive grade analyzed by Fisher’s unilateral
exact test.Statistical analysis was not done for clinical signs, necropsy findings or urinalysis data.
For the other data, the value obtained for each animal or litter mean was regarded as one sample, and the homogeneity of variance for each group was examined first by Bartlett’s test. Based on the results, the data were subjected to one-way analysis of variance or Kruskal-Wallis rank test, and the difference between the control group and each test article administration group was tested by Dunnett’s method or Scheffé’s method, if
significance was observed between groups. The levels of statistical significance were 5% and 1%.

Reproductive indices:

Copulation index [(number of copulated pairs / number of mated pairs) × 100], fertility index [(number of pregnant animals / number of copulated pairs) × 100], the number of days from the start of mating to the day copulation was confirmed.
The length of the gestation period (the number of days from day 0 of gestation and the day of delivery) was calculated and the gestation index
[(number of pregnant females with pups alive / number of pregnant females) × 100] calculated for each group.

Offspring viability indices:

Litter size (liveborn and stillborn pups) was counted on day 0 of lactation, and the delivery index [(number of pups born / number of implantation
sites) × 100] and the birth index [(number of pups alive on day 0 / number of implantation sites) × 100] were calculated. In addition, pups were
observed for the presence or absence of external malformation and sex, and the sex ratio of liveborn pups [(number of male pups born alive /
number of female pups born alive) × 100] calculated.
The number of pups that died was counted every day and the live birth index [(number of pups alive on day 0 / number of pups born) × 100] and the viability index on day 4 after birth [(number of pups alive on day 4 of lactation / number of pups alive on day 0 of lactation) × 100] were calculated.

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
For both males and females, there were neither deaths nor moribund animals in any dose group.

MALES: Two males showed decreased locomotor activity and 1 male showed transient salivation after dosing during the early administration period in the 60 mg/kg group, and all males showed transient salivation after dosing and decreased locomotor activity throughout the administration period, 9 males showed abdominal or lateral position, 2 males showed incomplete eyelid opening and 1 male showed leaning position during the early administration period in the 300 mg/kg group.

FEMALES: At the top dose of 300 mg/kg, all females showed transient salivation, decreased locomotor activity, abdominal or lateral position and 10 females showed staggering gait largely throughout the administration period, and 7 females showed incomplete eyelid opening and 3 females showed leaning position from the start to the middle of the administration period. These changes had disappeared by the time of administration of the next day.

There were no abnormalities in general condition in males or females in the control group or 12 mg/kg group or in females in the
60 mg/kg group.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): no treatment related effects observed

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): no treatment related effects observed

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): no treatment related effects observed

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): no treatment related effects observed

ORGAN WEIGHTS (PARENTAL ANIMALS): Comparison between the 300 mg/kg group and control group showed a small but significant increase
(p<0.05) in the liver weight body weight ratio. In the other organs, there were no significant differences in organ weight between the control group and any test article administration group.

GROSS PATHOLOGY (PARENTAL ANIMALS): no treatment related effects observed

HISTOPATHOLOGY (PARENTAL ANIMALS):
Males - Minimal centrilobular hypertrophy of hepatocytes was observed in the 300 mg/kg group and its incidence was significantly (p<0.01) higher than in the control group.

OTHER FINDINGS (PARENTAL ANIMALS)

Dose descriptor:

NOEL

Remarks:

Reproductive effects

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested: no effects from administration of the test article in any parameter measured.

Dose descriptor:

NOEL

Remarks:

General toxicity

Effect level:

12 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No effects seen at this dose

Dose descriptor:

NOEL

Remarks:

General toxicity

Effect level:

60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects seen at this dose

Dose descriptor:

LOEL

Remarks:

General toxicity

Effect level:

60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: 60 mg/kg - A decrease in locomotor activity was observed at this dose during the initial administration period in a small number of males. 300 mg/kg - Transient salivation, decreased activity and incomplete eyelid opening.

Dose descriptor:

LOEL

Remarks:

General toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Transient salivation, decreased activity and incomplete eyelid opening, staggering gait

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

VIABILITY (OFFSPRING): no treatment related effects observed

CLINICAL SIGNS (OFFSPRING): no treatment related effects observed

BODY WEIGHT (OFFSPRING): no treatment related effects observed

Dose descriptor:

NOEL

Remarks:

Pups

Generation:

F1

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were seen in pups

Reproductive effects observed:

not specified

Conclusions:

There were no adverse reproductive effects reported at any dose.
There were no effects to body weight, heamatology parametersl, blood chemistry, organ pathologies.
Clinical signs of transient salivation, decreased locomotor activity and staggering gait were observed at the top dose of 300 mg/kg (females) and at 60 mg/kg (males).
There were no adverse observations in the pups.
The NOEL for general toxicity is based on clinical signs only, as observed in parent animals.
the NOEL for reproductive is set at the top dose tested but could be higher.

Executive summary:

In a study report provided by the Japanese Ministry of Health, a combined repeat dose and reproductivescreening study on o-sec butyl phenol (OSBP) is described, as similar to the principles of an OECD guideline 422 study. Crj:CD(SD) rats (n=13 males and n=13 females per dose group) were administered with OSBP in corn oil, by oral gavage, at doses of 300, 60, 12 and 0 mg/kg. There were no changes in parent animals that were thought to be caused by administration of the test article in the results of body weight measurement, food consumption measurement, hematological examination, blood chemistry examination, urinalysis, necropsy or histopathological examination or reproductive effects. There were no adverse observations in pups.

Clinical observations that were thought to be caused by administration of the test article were observed in both males and females in the 300 mg/kg groups. These effects were transient salivation and decreased locomotor activity and (in females) staggering gait. Some clinical signs of transient salivation and decreased acitivity were observed in some males in the 60 mg/kg group. No adverse clinical effects were seen at the 12 mg/kg dose.

It is considered that the no observed effect level (NOEL) for reproductive effects of o-sec-butylphenol is >300 mg/kg/day (ie greater than the top dose tested) for both males and females under the conditions of this study. This substance is not a reproductive toxicant.

It is considered that the no observed effect level (NOEL) for general toxicity of o-sec-butylphenol is 12 mg/kg/day (males) and 60 mg/kg day (females) under the conditions of this study.

It is expected that o-tert butyl phenol with behave similarly to o-sec butyl phenol, and that OTBP would not be a reproductive toxicant.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 2. The study is well documented and follows the principles of OECD guideline 422. However, GLP compliance is not stated.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There are no reproduction studies available for o-tert amylphenol. There is a reproduction screening study available for the read across substance o-sec butylphenol.

In a study report provided by the Japanese Ministry of Health, a combined repeat dose and reproductive screening study on o-sec butyl phenol is described, performed as similar to the principles of an OECD guideline 422 study. Crj:CD(SD) rats (n=13 males and n=13 females per dose group) were administered the substance in corn oil, by oral gavage, at doses of 300, 60, 12 and 0 mg/kg. There were no changes in parent animals that were thought to be caused by administration of the test article in the results of body weight measurement, food consumption measurement, hematological examination, blood chemistry examination, urinalysis, necropsy or histopathological examination or any reproductive parameters. There were no adverse observations in pups.

The NOEL for reproductive effects is considered to be >300 mg/kg (i.e. greater than the top dose tested in this study).

Short description of key information:
A study report provided by the Japanese Ministry of Health, describing a combined repeat dose and reproductive screening study on the read across substance o-sec butyl phenol.

Justification for selection of Effect on fertility via oral route:
The only study available to address this endpoint is a study on o-sec butyl phenol (a read-across analogue of o-tert amylphenol) supplied by the Japanese Ministry of Health.

Effects on developmental toxicity

Description of key information

OECD guideline 414 study on read across substance, o-tert butyl phenol

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 2016 - January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy
- Age at study initiation: females 9 weeks, (males at least 14 weeks)
- Weight at study initiation: females 196-218 g; (males 322-355g)
- Fasting period before study: no
- Housing: No more than 5 of one sex to a cage, in polysulfon cages (59.5x38x20cm), nesting material provided inside suitable bedding bags, additionally suitable nesting material was provided as necessary. Nesting material was changed at least 3 times a week. During mating period, 1 male to 1 female in clear polysulfone cages (42.5x26.6x18.5cm) with a stainless steel mesh lid and floor, each cage tray held absorbent material which was inspected and changed daily
- Diet: commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Settimo Milanese, Italy) ad libitum
- Water: drinking water via water bottles ad libitum
- Acclimation period: approximately 2 weeks before the start of the treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 2016-02-02 To: 2016-03-04

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly intervals
- Mixing appropriate amounts with: powdered rodent diet (4RF21 Mucedola s.r.l., Settimo Milanese (MI), Italy) by initial preparation of a pre-mix followed by dilution with further quantities of diet and mixing
- Storage temperature of food: not mentioned

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated by the Analytical Chemistry Department at RTC according to RTC SOPs (RTC Study No. A1763) and the test item was found to be stable at room temperature for 8 days at the concentrations of 100 and 10000 ppm. Before treatment, analysis was performed to confirm that the method was suitable and the proposed formulation procedure was acceptable. Samples of the formulations prepared during the study (Week 1 and Week 3) were also analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in RTC SOPs for diet (80-120% for concentration and CV < 10% for homogeneity). Chemical analysis was carried out by the Analytical Chemistry Department at RTC.

Details on mating procedure:

Females were paired one to one male rat in the home cage of the male and left overnight. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of mating was made. The day of mating, as judged by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum). Full mating records were maintained.

Duration of treatment / exposure:

days 6 to 19 post coitum

Duration of test:

up to day 20 of gestation

Dose / conc.:

200 ppm (nominal)

Dose / conc.:

1 000 ppm (nominal)

Dose / conc.:

5 000 ppm (nominal)

Dose / conc.:

17.7 mg/kg bw/day (actual dose received)

Remarks:

see Table 1 below (Any other information on materials and methods incl. tables)

Dose / conc.:

89.51 mg/kg bw/day (actual dose received)

Remarks:

see Table 1 below (Any other information on materials and methods incl. tables)

Dose / conc.:

364.29 mg/kg bw/day (actual dose received)

Remarks:

see Table 1 below (Any other information on materials and methods incl. tables)

No. of animals per sex per dose:

24 mated females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were in agreement with the Sponsor based on a previous study in rats (RTC Study No.: X0310EXT)
Study design of this preliminary study:
Three groups, each of 6 mated female Sprague Dawley SD rats, received the test item in the diet (Mucedola, 4RF21) at the fixed inclusion levels of 200, 1000 and 5000 ppm (in terms of test item as supplied) during the gestation period, starting from Day 6 through Day 19 post coitum. A fourth similarly constituted group received the untreated diet and acted as a control. Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, uterus weight, foetal weight and sex were recorded. All foetuses were examined for external abnormalities.
Results of this preliminary study:
No clinical signs were noted in treated females and no differences of toxicological relevance were observed in bodyweight and bodyweight gain compared to controls. Food consumption was strongly reduced in the first days of administration in the high dose group compared to the control group, but an increment was detected in the following days. A moderate reduction in the absolute weight gain was detected in all treated groups, although without a dose-relation. Gravid uterus weight, litter data and macroscopic observations were not affected by treatment and no findings were detected at the external examination of foetuses. The calculated mean daily achieved dosages for the 14 days of treatment were approximately 16, 78 and 360 mg/kg/day. On the basis of the above results, the inclusion level of 5000 ppm is considered the NOAEL (No Observed Adverse Effect Level) in this study, corresponding to the achieved dosage of 360 mg/kg body weight/day of the test item.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each animal was observed daily, for mortality all animals were checked early in the morning and again in the afternoon, at weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: during the dosing period an additional observation for signs of reaction to treatment was performed at approx. 1,5-2 h after dosing


BODY WEIGHT: Yes
- Time schedule for examinations: all animals were weighed an Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes, measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting Day 0 post coitum
- Food consumption for each animal determined and mean daily diet consumption calculated: Yes, as g food/animal/day per cage
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, achieved dosage = ppm x food consumption / body weight

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 (carbon dioxide)
- Organs examined: necropsy (including examination of the external surface and orifices), changes were noted and the abnormalities preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol)

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: number, sex and weight of all live foetuses, number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing), gross evaluation of placentae, uteri or individual uterine horns without visible implantations were immersed in a 20 % solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation

Fetal examinations:

- External examinations: Yes: all live foetuses
- Soft tissue examinations: Yes: approx. half per litter
- Skeletal examinations: Yes: approx. half per litter
- Head examinations: Yes

Statistics:

For continuous variables the significance of the differences amongst group means was assessed by Dunnett's test (if group variances were homogeneous) or a modified t-test (if group variances were inhomogeneous).
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.

Indices:

Pre-implantation loss was calculated as a percentage from the formula: (no. of corpora lutea - no. of implantations) x 100/no. of corpora lutea
Post-implantation loss was calculated as a percentage from the formula: (no. of implantations - no. of live young) x 100/no. of implantations
Total implantation loss was calculated as a percentage from the formula: (no. of corpora lutea - no. of live young) x 100/no. of corpora lutea
Sex ratios of the foetuses were calculated as the percentage of males per litter
All derived values (e.g., means, percentages, ratios) were first calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decreases in body weight were detected in the high dose group starting from Day 9 post coitum until termination. The changes were up to 8%.
At body weight gain, a statistically significant decrease was noted on Days 9 and 20 post coitum in the high dose group when compared to control. These statistically decreases were of -105 % and -31% on Days 9 and 20 post coitum, respectively. For details see Tables 3 and 4 below (Any other information on results incl. tables).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significant decrease of approximately -50% was detected on Day 9 post coitum in females of the high dose group. A trend of recovery was noted in the subsequent days but a statistically significant decrease was still noted at the end of the study when the difference was -15% compared to the control group. For details see Table 5 below (Any other information on results incl. tables).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant decrease in uterus weight (-15%) was observed in females of the high dose group compared to controls. For details see Table 6 below (Any other information on results incl. tables).

Gross pathological findings:

no effects observed

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

An increased incidence in the number of early intrauterine deaths was observed in females of the high dose group compared to the control. As a consequence, the total number of viable foetuses was decreased. For details see Table 7 below (Any other information on results incl. tables).

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Post-implantation loss was observed in females of the high dose group. For details see Table 7 below (Any other information on results incl. tables).

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

89.51 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

In the high dose group, the total number of viable foetuses was decreased (approximately -14%). For details see Table 7 below (Any other information on results incl. tables).

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Litter size and litter weight was decreased in the high dose group (approximately -14%, resp. -16%). For details see Table 7 below (Any other information on results incl. tables).

External malformations:

no effects observed

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Two high dose foetuses, from two different dams, showed malformations. One foetus from dam no. 151 showed fused ribs and one foetus from dam no. 191 showed multiple alterations such as unossified ischium, pubis and basisphenoid and ulna and radius misshaped. Also these foetuses were small (foetal weight < 2.7 g), with a foetal weight of 1.90 g and 1.81 g, respectively. The other changes noted were comparable between groups. For details see Table 10 below (Any other information on results incl. tables).

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

In the high dose group two foetuses of two different dams (animal nos. 183 and 191) showed the pelvic region of the kidney extremely enlarged, unilaterally and bilaterally, respectively. One of these foetuses, from dam no. 191, also showed extreme enlargement of the ureters (bilateral). These alterations were classified as malformations. Minor alterations, classified as variations or anomalies, related to kidneys pelvi or ureters were also observed in the high dose group with severity from slight to moderate. The incidence, in terms of litters affected, of these findings reached 70% for ureters enlarged compared to 13% of the controls and 39% for pelvic dilatation compared to 4% of the control. An increased incidence in displaced testes (48% in the high dose group compared to 26% of the control group, in terms of litters affected) was also observed. The incidences of these findings were above the background range, but the maternal toxicity observed probably indicates that they may be secondary to maternal stress. For details see Table 11 below (Any other information on results incl. tables).

Key result

Dose descriptor:

NOAEL

Effect level:

89.51 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

reduction in number of live offspring

changes in litter size and weights

skeletal malformations

visceral malformations

Remarks on result:

other: Developmental effects were found only in maternal toxic dosage.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

364.29 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Table 2: Fate of females - group incidence

	Negative control group	Low dose	Medium dose	High dose
	0 ppm	200 ppm	1000 ppm	5000 ppm
Initial group size	24	24	24	24
Not pregnant	0	0	0	1
Total Resorption	1	0	0	0
Unilateral implantation	0	0	0	1
With live foetuses at gestation Day 20	23	24	24	23

Table 3: Body weight of females (g) with live foetuses at gestation day 20 - group mean data

	Day of phase
		0^G	3	6^D	9	12	15	18	20
Negative control (0 ppm)	(n)	23	23	23	23	23	23	23	23
	mean	238.54	254.60	267.42	279.21	296.00	318.63	364.90	398.39
	SD	8.56	8.96	9.50	9.28	9.29	10.66	11.33	14.82
Low dose (200 ppm)	(n)	24	24	24	24	24	24	24	24
	mean	235.97	251.91	265.88	275.97	293.40	313.23	360.77	395.57
	SD	11.94	9.57	10.22	10.94	11.73	12.49	13.46	15.70
Medium dose (1000 ppm)	(n)	24	24	24	24	24	24	24	24
	mean	238.64	256.19	268.20	278.69	293.49	315.21	362.03	395.84
	SD	11.21	9.70	12.26	10.62	11.31	12.28	13.89	17.39
High dose (5000 ppm)	(n)	23	23	23	23	23	23	23	23
	mean	235.06	251.52	262.78	262.14*	284.58*	303.57*	344.40*	367.59*
	SD	9.10	8.00	9.26	10.81	12.83	12.18	15.20	18.97

 ^G= Gestation phase;^D= Dosing/gestation phase; * = mean value of group is significantly different from control at p<0.05 (Dunnett's test)

 Table 4: Body weight gain per day^O(g) of females with live foetuses at gestation day 20 - group mean data

	Day of phase
		3^G	6^D	9	12	15	18	20
Negative control (0 ppm)	(n)	23	23	23	23	23	23	23
	mean	5.355	4.275	3.928	5.596	7.534	15.434	16.745
	SD	2.071	1.584	1.376	1.172	1.782	2.128	2.910
Low dose (200 ppm)	(n)	24	24	24	24	24	24	24
	mean	5.313	4.656	3.362	5.811	6.608	15.849	17.398
	SD	1.717	1.204	1.741	1.467	2.425	2.632	3.396
Medium dose (1000 ppm)	(n)	24	24	24	24	24	24	24
	mean	5.847	4.003	3.498	4.934	7.239	15.607	16.906
	SD	1.961	1.972	1.760	1.533	1.807	3.145	3.176
High dose (5000 ppm)	(n)	23	23	23	23	23	23	23
	mean	5.489	3.752	-0.214*	7.481 **($)	6.629	13.610	11.595*
	SD	1.56	1.209	2.119	2.347	3.853	3.797	3.586

^O= mean daily body weight gain over the previous period;^G= Gestation phase;^D= Dosing/gestation phase

 * = mean value of group is significantly different from control at p<0.05

 ** = mean value of group is significantly different from control at p<0.01

 Statistical analysis: Dunnett's test (group variances are homogeneous); $ = Modified t-test (group variances are inhomogeneous)

Table 5: Food consumption^O(g/animals/day) of females - group mean data

	Day of phase
		3^G	6^D	9	12	15	18	20
Negative control (0 ppm)	(n)	9	9	9	9	9	9	9
	mean	26.87	26.48	28.63	30.21	31.03	33.06	30.40
	SD	3.48	5.01	5.12	4.74	4.48	3.24	6.34
Low dose (200 ppm)	(n)	10	10	10	10	10	10	10
	mean	25.01	26.24	26.53	28.13	27.74	30.13	31.01
	SD	2.45	1.76	4.82	2.87	2.48	6.02	3.50
Medium dose (1000 ppm)	(n)	10	10	10	10	10	10	10
	mean	25.45	27.81	26.02	29.93	28.79	30.70	30.11
	SD	3.93	6.98	5.17	9.69	4.39	4.17	5.94
High dose (5000 ppm)	(n)	9	9	9	9	9	9	9
	mean	23.56	24.66	13.99*	22.74**($)	24.03*	27.19**($)	25.84
	SD	3.24	3.39	4.11	3.41	2.24	1.63	2.99

^O= food consumed over the previous phase starting from allocation;^G= Gestation phase;^D= Dosing/gestation phase

 * = mean value of group is significantly different from control at p<0.05

 ** = mean value of group is significantly different from control at p<0.01

 Statistical analysis: Dunnett's test (group variances are homogeneous); $ = Modified t-test (group variances are inhomogeneous)

Table 6: Terminal body weight, uterus weight and absolute weight gain of females with live foetuses - group mean data

		Terminal body weight (g)		Gravid uterus weight (g)		Absolute weight gain# (g)
Negative control (0 ppm)	(n)	23		23		23
	mean	394.97		85.18		71.25
	SD	15.75		10.99		7.36
Low dose (200 ppm)	(n)	24		24		24
	mean	390.97		84.33		70.65
	SD	16.36		8.57		13.05
Medium dose (1000 ppm)	(n)	24		24		24
	mean	391.28		83.85		68.79
	SD	17.84		10.16		9.05
High dose (5000 ppm)	(n)	23		23		23
	mean	362.08*		72.17*		54.87*
	SD	19.44		16.19		6.98

# = body weight at necropsy minus gravid uterine wt., minus body wt. at Day 0 of pregnancy

 * = statistically significantly different from control group value at p<0.05

Table 7: Litter data and sex ratios - group mean data

				Uterine deaths			Viable young			%	Implantation loss (%)
		Corpora Lutea	Implantations	Early	Late	Total	Total	M	F	Males	Pre	Post	Total	Litter weight (g)	Mean foetal weight (g)
Negative control (0 ppm)	(n)	23	23	23	23	23	23	23	23	23	23	23	23	23	23
	mean	16.22	16.04	0.78	0.00	0.78	15.26	7.43	7.83	47.96	1.26	4.74	5.98	56.05	3.68
	SD	1.88	2.14	1.28	0.00	1.28	2.28	2.69	1.95	12.57	3.66	7.43	7.70	8.35	0.21
Low dose (200 ppm)	(n)	24	24	24	24	24	24	24	24	24	24	24	24	24	24
	mean	15.79	15.54	0.54	0.04	0.58	14.96	7.13	7.83	47.84	1.62	3.85	5.42	55.30	3.71
	SD	1.74	1.84	0.93	0.20	0.93	2.07	1.83	2.06	10.70	3.42	6.42	6.70	6.99	0.26
Medium dose (1000 ppm)	(n)	24	24	24	24	24	24	24	24	24	24	24	24	24	24
	mean	15.67	15.58	0.42	0.00	0.42	15.17	6.96	8.21	45.85	0.59	2.68	3.27	55.71	3.69
	SD	2.14	2.21	0.78	0.00	0.78	2.32	2.33	2.34	12.97	2.04	4.96	5.03	7.89	0.26
High dose (5000 ppm)	(n)	23	23	23	23	23	23	23	23	23	23	23	23	23	23
	mean	14.57	14.52	1.39	0.00	1.39	13.13	6.09	7.04	46.55	0.52	8.93	9.11	47.05*	3.61
	SD	2.76	2.71	3.16	0.00	3.16	3.48	2.37	2.51	14.62	1.74	17.82	18.04	12.28	0.25

= statistically significantly different from control group value at p<0.05

Table 8: Macroscopic observations - group incidence

	Females
	Negative control group	Low dose	Medium dose	High dose
	0 ppm	200 ppm	1000 ppm	5000 ppm
Number in group	24	24	24	24
Uterus: abnormal content	0	0	0	1
Not pregnant	0	0	0	1
Total Resorption	1	0	0	0
Unilateral implantation	0	0	0	1
Whole animal: No abnormalities detected	23	24	24	22

Table 9: External examination of foetuses - group incidence

					No. foetuses			No. litters
Group	Organ	Cat	Observation(s)		Observed	Affected	%	Observed	Affected	%
Negative control (0 ppm)	Whole foetus		No abnormalities detected		351	149	99.43	23	-	-
		AN	Small		351	2	0.57	23	2	8.70
Low dose (200 ppm)	Whole foetus		No abnormalities detected		359	355	98.89	24	-	-
		AN	Small		359	4	1.11	24	4	16.67
Medium dose (1000 ppm)	Whole foetus		No abnormalities detected		364	359	98.63	24	-	-
		AN	Small		364	5	1.37	24	3	12.50
High dose (5000 ppm)	Whole foetus		No abnormalities detected		302	297	98.34	23	-	-
		AN	Small		302	5	1.66	24	3	13.04

Table 10: Skleletal examination of foetuses - group incidence

						No. foetuses			No. dams
Group	Organ	Cat	Observation(s)			Observed	Affected	%	Observed	Affected	%
Negative control (0 ppm)	Forepaw(s)	AN	Metacarpal(s) no ossification 4th			181	27	14.92	23	9	39.13
	Forepaw(s)	AN	Metacarpal(s) no ossification			181	1	0.55	23	1	4.35
	Hindpaw(s)	AN	Metatarsal(s) no ossification			181	2	1.10	23	1	4.35
	Hindpaw(s)	VA	Metatarsal(s) incomplete ossification 4th			181	1	0.55	23	1	4.35
	Ribs	VA	Rudimentary 14th			181	12	6.63	23	7	30.43
	Skull	AN	Temporal incomplete ossification			181	26	14.36	23	15	65.22
	Skull	VA	Interparietal incomplete ossification			181	1	0.55	23	1	4.35
	Skull	VA	Parietal incomplete ossification			181	1	0.55	23	1	4.35
	Sternebrae	AN	No ossification			181	2	1.10	23	2	8.70
	Sternebrae	AN	Asymmetrical ossification			181	13	7.18	23	9	39.13
	Sternebrae	AN	Asymmetrical ossification 5th			181	22	12.15	23	8	34.78
	Sternebrae	AN	No ossification 6th			181	2	1.10	23	1	4.35
	Sternebrae	VA	No ossification 5th			181	8	4.42	23	5	21.74
	Sternebrae	VA	Incomplete ossification 6th			181	40	22.10	23	13	56.52
	Sternebrae	VA	Incomplete ossification 5th			181	34	18.78	23	14	60.87
	Sternebrae	VA	Incomplete ossification			181	10	5.52	23	8	34.78
	Thoracic vertebrae	VA	Centrum incomplete ossification			181	11	6.08	23	7	30.43
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped			181	4	2.21	23	3	13.04
	Thoracic vertebrae	VA	Centrum dumb-bell shaped			181	12	6.63	23	10	43.48
Low dose (200 ppm)	Forepaw(s)	AN	Metacarptal(s) no ossification 4th			186	33	17.74	24	10	41.67
	Hindpaw(s)	AN	Metatarsal(s) no ossification			186	2	1.08	24	1	4.17
	Hindpaw(s)	AN	Metatarsal(s) no ossification 4th			186	1	0.54	24	1	4.17
	Hindpaw(s)	VA	Metatarsal(s) incomplete ossification 4th			186	1	0.54	24	1	4.17
	Lumbar vertebrae	AN	Centrum asymmetrical dumb-bell shaped			186	1	0.54	24	1	4.17
	Pelvic girdle	AN	Pubis incomplete ossification			186	2	1.08	24	2	8.33
	Ribs	VA	Rudimentary 14th			186	18	9.68	24	9	37.50
	Ribs	VA	Short 14th			186	1	0.54	24	1	4.17
	Sacral vertebrae	AN	Arch(es) incomplete ossification			186	1	0.54	24	1	4.17
	Sacral vertebrae	VA	Centrum incomplete ossification			186	1	0.54	24	1	4.17
	Skull	AN	Temporal incomplete ossification			186	32	17.20	24	11	45.83
	Sternebrae	AN	No ossification			186	2	1.08	24	2	8.33
	Sternebrae	AN	Asymmetrical ossification 5th			186	20	10.75	24	8	33.33
	Sternebrae	AN	Asymmetrical ossification			186	16	8.60	24	6	25.00
	Sternebrae	AN	No ossification 6th			186	3	1.61	24	3	12.50
	Sternebrae	VA	Incomplete ossification 6th			186	35	18.82	24	12	50.00
	Sternebrae	VA	No ossification 5th			186	7	3.76	24	4	16.67
	Sternebrae	VA	Incomplete ossification 5th			186	31	16.67	24	15	62.50
	Sternebrae	VA	Incomplete ossification			186	10	5.38	24	7	29.17
	Thoracic vertebrae	AN	Centrum no ossification			186	2	1.08	24	2	8.33
	Thoracic vertebrae	AN	Centrum bipartite			186	1	0.54	24	1	4.17
	Thoracic vertebrae	VA	Centrum incomplete ossification			186	7	3.76	24	5	20.83
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped			186	1	0.54	24	1	4.17
	Thoracic vertebrae	VA	Centrum dumb-bell shaped			186	20	10.75	24	12	50.00
Medium dose (1000 ppm)	Forepaw(s)	AN	Metacarpal(s) no ossification 4th			188	30	15.96	24	12	50.00
	Forepaw(s)	AN	Metacarpal(s) no ossification			188	1	0.53	24	1	4.17
	Hindpaw(s)	AN	Metatarsal(s) no ossification			188	1	0.53	24	1	4.17
	Lumbar vertebrae	AN	Centrum dumb-bell shaped			188	1	0.53	24	1	4.17
	Lumbar vertebrae	VA	Centrum incomplete ossification			188	1	0.53	24	1	4.17
	Pelvic girdle	AN	Pubis incomplete ossification			188	2	1.06	24	2	8.33
	Ribs	AN	Wavy			188	1	0.53	24	1	4.17
	Ribs	VA	Rudimentary 14th			188	19	10.11	24	10	41.67
	Sacral vertebrae	AN	Arch(es) incomplete ossification			188	1	0.53	24	1	4.17
	Skull	AN	Frontal incomplete ossification			188	1	0.53	24	1	4.17
	Skull	AN	Temporal incomplete ossification			188	36	19.15	24	17	70.83
	Skull	VA	Interparietal incomplete ossification			188	2	1.06	24	2	8.33
	Sternebrae	AN	Bipartite and asymm ossification			188	1	0.53	24	1	4.17
	Sternebrae	AN	Asymmetrical ossification			188	13	6.91	24	8	33.33
	Sternebrae	AN	No ossification 6th			188	5	2.66	24	4	16.67
	Sternebrae	AN	No ossification			188	4	2.13	24	3	12.50
	Sternebrae	AN	Asymmetrical ossification 5th			188	22	11.70	24	11	45.83
	Sternebrae	VA	Incomplete ossification 6th			188	33	17.55	24	13	54.17
	Sternebrae	VA	Incomplete ossification			188	16	8.51	24	11	45.82
	Sternebrae	VA	No ossification 5th			188	5	2.66	24	4	16.67
	Sternebrae	VA	Incomplete ossification 5th			188	32	17.02	24	16	66.67
	Thoracic vertebrae	AN	Centrum bipartite			188	2	1.06	24	2	8.33
	Thoracic vertebrae	AN	Centrum asymmetrical ossification			188	1	0.53	24	1	4.17
	Thoracic vertebrae	AN	Centrum no ossification			188	4	2.13	24	3	12.50
	Thoracic vertebrae	VA	Centrum dumb-bell shaped			188	6	3.19	24	5	20.83
	Thoracic vertebrae	VA	Centrum incomplete ossification			188	8	4.26	24	6	25.00
High dose (5000 pm)	Forelimb(s)	AN	Ulna incomplete ossification			157	1	0.64	23	1	4.35
	Forelimb(s)	AN	Omerus incomplete ossification			157	1	0.64	23	1	4.35
	Forelimb(s)	AN	Radius incomplete ossification			157	1	0.64	23	1	4.35
	Forelimb(s)	MA	Ulna mishaped			157	1	0.64	23	1	4.35
	Forelimb(s)	MA	Radius mishaped			157	1	0.64	23	1	4.35
	Forepaw(s)	AN	Metacarpal(s) no ossification			157	1	0.64	23	1	4.35
	Forepaw(s)	AN	Metacarpal(s) no ossification 4th			157	35	22.29	23	12	52.17
	Forepaw(s)	AN	Metacarpal(s) incomplete ossification			157	1	0.64	23	1	4.35
	Hindlimb(s)	AN	Femur incomlete ossification			157	1	0.64	23	1	4.35
	Hindlimb(s)	AN	Fibula incomplete ossification			157	1	0.64	23	1	4.35
	Hindlimb(s)	AN	Tibia incomplete ossification			157	1	0.64	23	1	4.35
	Hindpaw(s)	AN	Metatarsal(s) incomplete ossification			157	1	0.64	23	1	4.35
	Hindpaw(s)	AN	Metatarsal(s) no ossification 4th			157	1	0.64	23	1	4.35
	Lumbar vertebrae	AN	Arch(es) incomplete ossification			157	1	0.64	23	1	4.35
	Lumbar vertebrae	AN	Centrum no ossification			157	1	0.64	23	1	4.35
	Lumbar vertebrae	VA	Centrum incomplete ossification			157	2	1.27	23	1	4.35
	Pectoral girdle	AN	Clavicle incomplete ossification			157	1	0.64	23	1	4.35
	Pectoral girdle	AN	Scapula incomplete ossification			157	1	0.64	23	1	4.35
	Pelvic girdle	MA	Pubis no ossification			157	1	0.64	23	1	4.35
	Pelvic girdle	MA	Ischium no ossification			157	1	0.64	23	1	4.35
	Ribs	AN	Wavy			157	1	0.64	23	1	4.35
	Ribs	AN	Incomplete ossification			157	1	0.64	23	1	4.35
	Ribs	MA	Fused			157	1	0.64	23	1	4.35
	Ribs	VA	Rudimentary 14th			157	24	15.29	23	11	47.83
	Ribs	VA	Short 14th			157	1	0.64	23	1	4.35
	Ribs	VA	14 ribs			157	1	0.64	23	1	4.35
	Sacral vertebrae	AN	Arch(es) incomplete ossification			157	4	2.55	23	4	17.37
	Sacral vertebrae	AN	Arch(es) no ossification			157	1	0.64	23	1	4.35
	Sacral vertebrae	AN	Centrum no ossification			157	1	0.64	23	1	4.35
	Skull	AN	Mandible incomplete ossification			157	1	0.64	23	1	4.35
	Skull	AN	Temporal incomplete ossification			157	19	12.10	23	10	43.48
	Skull	AN	General incomplete ossification			157	1	0.64	23	1	4.35
	Skull	MA	Basisphenoid no ossification			157	1	0.64	23	1	4.35
	Skull	VA	Interparietal incomplete ossification			157	2	1.27	23	1	4.35
	Sternebrae	AN	Asymmetrical ossification 5th			157	13	8.28	23	9	39.13
	Sternebrae	AN	No ossification			157	6	3.82	23	3	13.04
	Sternebrae	AN	No ossification 6th			157	2	1.27	23	2	8.70
	Sternebrae	AN	Rudimentary 5th			157	2	1.27	23	2	8.70
	Sternebrae	AN	Asymmetrical ossification			157	10	6.37	23	5	21.76
	Sternebrae	VA	Incomplete ossification 5th			157	29	18.47	23	14	60.87
	Sternebrae	VA	No ossification 5th			157	9	5.73	23	6	26.09
	Sternebrae	VA	Incomplete ossification 6th			157	23	14.64	23	12	52.17
	Sternebrae	VA	Incoomplete ossification			157	8	5.10	23	5	21.74
	Thoracic vertebrae	AN	Arch(es) incomplete ossification			157	1	0.64	23	1	4.35
	Thoracic vertebrae	AN	Centrum no ossification			157	4	2.55	23	2	8.70
	Thoracic vertebrae	AN	Centrum bipartite			157	2	1.27	23	1	4.35
	Thoracic vertebrae	AN	Haemicentrum			157	2	1.27	23	1	4.35
	Thoracic vertebrae	AN	Centrum asymmetrical ossification			157	2	1.27	23	2	8.70
	Thoracic vertebrae	VA	Centrum dumb-bell shaped			157	16	10.19	23	11	47.83
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped			157	1	0.64	23	1	4.35
	Thoracic vertebrae	VA	Centrum incomplete ossification			157	9	5.73	23	6	26.09

Table 11: Visceral examination of foetuses - group incidence

						No. foetuses			No. litters
Group	Organ	Cat	Observation(s)			Observed	Affected	%	Observed	Affected	%
Negative control (0 ppm)	Abdomen	VA	Haemorrhagic			170	1	0.59	23	1	4.35
	Heart	AN	Atrium enlarged			170	13	7.65	23	8	34.78
	Kidneys	AN	Pelvic dilatation moderate			170	1	0.59	23	1	4.35
	Kidneys	AN	Ectopic			170	4	2.35	23	3	13.04
	Testis	AN	Displaced			170	6	3.53	23	6	26.09
	Ureter	AN	Kinked moderate			170	1	0.59	23	1	4.35
	Ureter	AN	Enlarged moderate			170	1	0.59	23	1	4.35
	Ureter	VA	Enlarged slight			170	2	1.18	23	2	8.70
	Whole foetuses	-	No abnormalities detected			170	146	85.88	23	-	-
Low dose (200 ppm)	Heart	AN	Atrium enlarged			173	4	2.31	24	3	12.50
	Kidneys	AN	Ectopic			173	2	1.16	24	2	8.33
	Kidneys	AN	Pelvic dilatation moderate			173	3	1.73	24	3	12.50
	Kidneys	VA	Pelvic dilatation slight			173	3	1.73	24	3	12.50
	Testis	AN	Displaced			173	3	1.73	24	3	12.50
	Ureter	AN	Enlarged moderate			173	4	2.31	24	3	12.50
	Ureter	VA	Enlarged slight			173	5	2.89	24	4	16.67
	Whole foetus	AN	Generalised oedema slight			173	4	2.31	24	3	12.50
	Whole foetuses	-	No abnormalities detected			173	149	86.13	24	-	-
Medium dose (1000 ppm)	Great vessels	VA	Innominate artery short			176	2	1.14	24	2	8.33
	Heart	AN	Atrium enlarged			176	2	1.14	24	2	8.33
	Kidneys	MA	Pelvic dilatation extreme			176	2	1.14	24	2	8.33
	Kidneys	VA	Pelvic dilatation slight			176	2	1.14	24	2	8.33
	Testis	AN	Displaced			176	5	2.84	24	5	20.83
	Ureter	AN	Enlarged moderate			176	2	1.14	24	2	8.33
	Ureter	MA	Enlarged extreme			176	2	1.14	24	2	8.33
	Ureter	VA	Enlarged slight			176	6	3.41	24	4	16.67
	Whole foetus	AN	Generalised oedema slight			176	3	1.70	24	3	12.50
	Whole foetuses	-	No abnormalities detected			176	156	88.64	24	-	-
High dose (5000 ppm)	Abdomen	VA	Haemorrhagic			145	8	5.52	23	3	13.04
	Head	AN	Haemorrhage			145	1	0.69	23	1	4.35
	Heart	AN	Atrium enlarged			145	5	3.45	23	4	17.39
	Kidneys	AN	Pelvic dilatation moderate			145	3	2.07	23	3	13.04
	Kidneys	MA	Pelvic dilatation extreme			145	2	1.38	23	2	8.70
	Kidneys	VA	Pelvic dilatation slight			145	8	5.52	23	6	26.09
	Testis	AN	Displaced			145	23	15.86	23	11	47.83
	Thoraric cavity	AN	Haemorrhage			145	1	0.69	23	1	4.35
	Ureter	AN	Enlarged moderate			145	7	4.83	23	6	26.09
	Ureter	MA	Enlarged extreme			145	1	0.69	23	1	4.365
	Ureter	VA	Enlarged slight			145	16	11.03	23	10	43.48
	Ureter	VA	Kinked slight			145	1	0.69	23	1	4.35
	Whole foetus	AN	Generalised oedema slight			145	1	0.69	23	1	4.35
	Whole foetuses	-	No abnormalities detected			145	94	64.83	23	-	-

Conclusions:

On the basis of the results obtained in this study, the inclusion level of 1000 ppm corresponding to the achieved dosage of 89.51 mg/kg body weight/day of O-T-BUTYLPHENOL was considered to be the NOAEL (No Observed Adverse Effect Level) for maternal toxicity and for developmental toxicity. Developmental effects were found only in maternal toxic dosage.

Executive summary:

The effects of O-T-BUTYLPHENOL were investigated after oral administration, via the diet, in female rats during pregnancy and on embryo-foetal development.

Three groups, each of 24 mated female Sprague Dawley SD rats, received the test item in the diet (Mucedola, 4RF21) at the fixed inclusion levels of 200, 1000 and 5000 ppm (in terms of test item as supplied) during the gestation period, starting from Day 6 through Day 19 post coitum. A fourth similarly constituted group received the untreated diet and acted as a control.

Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, uterus weight, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

The calculated mean achieved dosages for the treatment period from Day 6 to Day 19 post coitum were approximately 18, 90 and 364 mg/kg/day respectively in low, mid- and high dose groups, in terms of test item as supplied.

No animals died during the study. The number of females with live foetuses on gestation Day 20 was 23 in the control and high dose groups and 24 in the low and mid-dose groups.

No clinical signs were noted during the study and no signs of reaction to treatment were observed during the dosing period.

Statistically significant differences in the body weight were limited up to -8% during the study, between control and high dose females. On the contrary the differences (decrease) in body weight gain were more pronounced throughout the study and particularly evident on Day 9 post coitum.

A statistically significant decrease in food consumption was detected starting from Day 9 up to Day 18 post coitum in females of the high dose group. Although a trend of recovery was noted, food consumption of the high dose group was still lower than control group on Day 20 post coitum.

A statistically significant decrease in terminal body weight, uterus weight and absolute weight gain was observed in females of the high dose group compared to controls.

An increased incidence in the number of intrauterine deaths (early and total) and post and total implantation loss, expressed as percentage, was observed in females of the high dose group compared to the control. As a consequence, the total number of viable foetuses was decreased. Litter weight was also decreased.

Animals killed at termination did not show any relevant macroscopic changes.

No abnormalities were detected at the external examination of foetuses. A total of 16 small foetuses were detected; two out of 351 in the control group, four out of 359 in low dose group, five out of 364 in the mid-dose group and five out of 302 in the high dose group.

One small foetus of the high dose group showed multiple malformations at skeletal examination: unossified ischium, pubis and basisphenoid and ulna and radius misshaped. An additional small foetus in the same group showed fused ribs.

At visceral examination, 2 foetuses of the high dose group showed major alterations localised to the kidneys and ureters. Moreover, an increase incidence (in terms of litters affected) in minor alterations, classified as variations or anomalies, always related to kidneys or ureters, was also observed. The incidences of these findings were above the background range, but the maternal toxicity observed probably indicates that they may be secondary to maternal stress.

Taking into account that some alterations in the urinary system were also noted in the control group, the findings noted at the mid-dose level could be considered incidental.

On the basis of the results obtained in this study, the inclusion level of 1000 ppm corresponding to the achieved dosage of 89.51 mg/kg body weight/day of O-T-BUTYLPHENOL was considered to be the NOAEL for maternal toxicity and for developmental toxicity. Developmental effects were found only in maternal toxic dosage.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

89.51 mg/kg bw/day

Species:

rat

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

The effects of the read across substance, o-t-Butylphenol, were investigated after oral administration, via the diet, in female rats during pregnancy and on embryo-foetal development. Three groups, each of 24 mated female Sprague Dawley SD rats, received the test item in the diet at the fixed inclusion levels of 200, 1000 and 5000 ppm (calculated mean achieved dosages were 18, 90 and 364 mg/kg/day) during the gestation period, starting from Day 6 through Day 19 post coitum. A fourth similarly constituted group received the untreated diet and acted as a control.

Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, uterus weight, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

No animals died during the study. The number of females with live foetuses on gestation Day 20 was 23 in the control and high dose groups and 24 in the low and mid-dose groups. No clinical signs were noted during the study and no signs of reaction to treatment were observed during the dosing period. Statistically significant differences in the body weight were limited up to -8% during the study, between control and high dose females. On the contrary the differences (decrease) in body weight gain were more pronounced throughout the study and particularly evident on Day 9 post coitum. A statistically significant decrease in food consumption was detected starting from Day 9 up to Day 18 post coitum in females of the high dose group. Although a trend of recovery was noted, food consumption of the high dose group was still lower than control group on Day 20 post coitum. A statistically significant decrease in terminal body weight, uterus weight and absolute weight gain was observed in females of the high dose group compared to controls.

An increased incidence in the number of intrauterine deaths (early and total) and post and total implantation loss, expressed as percentage, was observed in females of the high dose group compared to the control. As a consequence, the total number of viable foetuses was decreased. Litter weight was also decreased. No abnormalities were detected at the external examination of foetuses. A total of 16 small foetuses were detected; two out of 351 in the control group, four out of 359 in low dose group, five out of 364 in the mid-dose group and five out of 302 in the high dose group. One small foetus of the high dose group showed multiple malformations at skeletal examination: unossified ischium, pubis and basisphenoid and ulna and radius misshaped. An additional small foetus in the same group showed fused ribs. At visceral examination, 2 foetuses of the high dose group showed major alterations localised to the kidneys and ureters. Moreover, an increase incidence (in terms of litters affected) in minor alterations, classified as variations or anomalies, always related to kidneys or ureters, was also observed. The incidences of these findings were above the background range, but the maternal toxicity observed probably indicates that they may be secondary to maternal stress. Taking into account that some alterations in the urinary system were also noted in the control group, the findings noted at the mid-dose level could be considered incidental.

On the basis of the results obtained in this study, the inclusion level of 1000 ppm corresponding to the achieved dosage of 89.51 mg/kg body weight/day of the read across substance, o-t-Butylphenol, was considered to be the NOAEL for maternal toxicity and for developmental toxicity. Developmental effects were found only in maternal toxic dosage.

Justification for classification or non-classification

Evidence from an OECD 422 Combined repeat dose and reproductive screening study on read-across analogues o-sec butyl phenol and an OECD 414 developmental study on the read across substance, o-t-Butylphenol, indicates that

o-tert amylphenol should not be classified as a reproductive toxicant.