Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 November 2016 to 02 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

Deviations:

yes

Remarks:

See "Any other information" for details

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guidelines, OPPTS 870.3550,

Version / remarks:

The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Limit test:

no

Justification for study design:

The purpose of this study was to evaluate the potential toxic effects of the test item when administered to rats by oral gavage for a minimum of 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.
Parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.
This study should provide part of a rational basis for toxicological risk assessment in man. The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item.

Test material

Test material information

Constituent 1

Reference substance name:

Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

EC Number:

701-003-6

Cas Number:

1454803-04-3

Molecular formula:

C20H39NO3 to C26H51NO3

IUPAC Name:

Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

Test material form:

liquid

Details on test material:

Identification: MLA-3202
Appearance: Clear amber-red liquid
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until 17 February 2019 (expiry date)
Purity/composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Amides, tallow, N,N-bis(2-hydroxypropyl)
CAS Number: 1454803-04-3
Test item handling No specific handling conditions required

Specific details on test material used for the study:

No further details specified in the study report

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

Test system: Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
Rationale: This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
Source F0: Charles River Deutschland, Sulzfeld, Germany.
Age at start F0-treatment: Approximately 11 weeks.
Number of F0-animals: 40 females and 40 males.
Acclimatization F0: At least 5 days prior to start of treatment.
Health inspection F0: At least upon receipt of the animals.
Randomization F0: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification F0: Earmark and tattoo.
Conditions: Environmental controls for the animal room were set to maintain 18 to 24 °C, a relative humidity of 40 to 70%, at least 10 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages
(MIII type, height 18 cm).
Lactation: Females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam.
General: Sterilized sawdust as bedding material (Lignocel S 8-15, JRS -J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water: Free access to tap-water.
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Vehicle: Water (Elix, Millipore S.A.S., Molsheim, France).
Rationale for vehicle: Based on trial formulations performed at Charles River Den Bosch.
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.
Appearance of formulations: Suspension (Groups 2-4).
Storage conditions: At room temperature.

Details on mating procedure:

Mating procedures
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated.
Detection of mating was not confirmed for animal nos. 48 and 50 which delivered live offspring. The mating date of these animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum. Detection of mating for animal nos. 42, 51 and 73 was confirmed later by evidence of sperm in the vaginal lavage after re-analysis of the lavages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (16 November 2016) according to a validated method (Test Facility Study No. 514869).
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

Duration of treatment / exposure:

Males were dosed for 29 days. Females that delivered were dosed for 42-46 days (most females) or 56 days (two females). Female nos. 41, 46 and 73 which failed to deliver healthy offspring were treated for 44, 53 and 39 days, respectively.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Details on study schedule:

Males were dosed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females that delivered were dosed for 42-46 days (most females) or 56 days (two females), i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 5 days after delivery up to and including the day before scheduled necropsy. Female nos. 41, 46 and 73 which failed to deliver healthy offspring were treated for 44, 53 and 39 days, respectively.
Routinely, females that are littering are left undisturbed. In this study, female nos. 42 (Group 1), 53 (Group 2) 71 and 79 (Group 4) were not treated for one day as they were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 animals per dose group (10 male/10 female)

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected based on the preliminary results (up to Day 17 of treatment) of a 28-day repeated dose toxicity study (Test Facility Study No. 514867). Treatment related clinical signs consisted of salivation in the mid and high dose groups for both males and females. Males showed a slightly decreased body weight gain at 1000 mg/kg. For the females a dose related increase in body weight gain was noted, statistically significant on Day 15 at 300 and 1000 mg/kg. Food consumption in these females appears unaffected by treatment.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

Mortality / Viability: At least twice daily.

Clinical signs: At least once daily after dosing from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals.
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.

Body weights: Males and females were weighed on the first day of exposure (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.

Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used to aid in confirmation of pregnancy. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females were examined for evidence of premature delivery. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
In female nos. 42, 48, 50 (Group 1), 51 Group 2) and 73 (Group 4) evidence of mating was obtained by palpation or by re-analysis of vaginal smears. Apparently, mating was overlooked in the assessment of the daily vaginal lavage.

Oestrous cyclicity (parental animals):

Not specified

Sperm parameters (parental animals):

Not specified

Litter observations:

Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective tables.
Body weights: Live pups were weighed on PND 1 and 4.
Sex: Sex was determined for all pups on PND 1 and 4.

Postmortem examinations (parental animals):

F0-generation - Pathology
F0-generation - Termination
The animals were not deprived of food overnight. All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.
Necropsy was conducted on the following days:
Condition Day of necropsy
Males Following completion of the mating period (a minimum of 28 days of dose administration).
Females which delivered PND 6-7.
Females which failed to deliver Post-coitum Day 25 (female no. 41 with evidence of mating) or 26 days after the last day of the mating period
(nos. 41,46) (female no. 46 without evidence of mating).
Female with total litter loss Within 24 hours of litter loss.
(no. 73)

F0-generation – Macroscopic Examination
After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
The numbers of former implantation sites and corpora lutea were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at Charles River Den Bosch using Ammoniumsulfidesolution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).
Samples of the following tissues and organs were collected from all animals and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
Tissues/organs Males Females
Identification marks: not processed Yes Yes
Cervix --- Yes
Clitoral gland --- Yes
Coagulation gland Yes ---
(Cowper’s glands) Yes ---
Epididymides Yes ---
Mammary gland area Yes Yes
(Glans penis) Yes ---
(Levator ani plus bulbocavernosus muscle complex (LABC)) Yes ---
Ovaries --- Yes
Preputial gland Yes ---
Pituitary gland Yes Yes
Prostate gland Yes ---
Seminal vesicles Yes ---
Testes Yes ---
Thyroid, including parathyroid if detectable Yes Yes
Uterus --- Yes
Vagina --- Yes
All gross lesions Yes Yes
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

F0-generation – Organ Weights
The following organ weights and terminal body weight were recorded from all males on the scheduled day of necropsy:
Epididymides
Testes
Absolute organ weights and organ to body weight ratios are reported.

F0-generation - Histotechnology
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

F0-generation – Histopathology
The following slides were examined by a pathologist:
• The testes, epididymides, ovaries, uterus, cervix and vagina of the animals of Groups 1 and 4.
• Additional slides of the testes of all males of Groups 1 and 4 and all males that failed to sire.
• All gross lesions of all animals (all dose groups).
• The reproductive organs2 of all males that failed to sire and all females that failed to deliver healthy pups.

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
A peer review on the histopathology data was performed by a second pathologist.

Postmortem examinations (offspring):

F1-generation Pups - Termination
Pups surviving to planned termination were killed by decapitation on Days 6 or 7 of lactation.

F1-generation pups - Macroscopic Examination
All pups were sexed and descriptions of all external abnormalities were recorded.
External abnormalities of pup 4 litter 44, pup 6 litter 62, pup 3 litter 69 and pup 9 litter 74 were collected and fixed in 10% buffered formalin at discretion of the Study Director.
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyse the data:
-If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
-The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
-The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:

Mating index (%) [Number of females mated/Number of females paired] x 100

Fertility index (%) [Number of pregnant females/Number of females paired] x 100

Conception index (%) [Number of pregnant females/Number of females mated] x 100

Gestation index (%) [Number of females with living pups on PND 1/Number of pregnant females] x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition.

Offspring viability indices:

For each group, the following calculations were performed:

Percentage live males at First Litter Check (%) [Number of live males pups at First Litter Check/Number of live pups at First Litter Check] x 100

Percentage live females at First Litter Check (%) [Number of live female pups at First Litter Check/Number of live pups at First Litter Check] x 100

Percentage of postnatal loss (%) [Number of dead pups before planned necropsy/Number of live pups at First Litter Check] x 100

Viability index (%) [Number of alive pups before planned necropsy/Number of pups born alive] x 100

Group mean values were calculated from individual litter values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs that were considered to be of toxicological relevance.
Salivation occurred after dosing in all males and females at 300 and 1000 mg/kg, generally starting in the first week of treatment, and in two males at 100 mg/kg starting in the fourth week of treatment. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its nature and severity (slight up to 300 mg/kg, slight or moderate at 1000 mg/kg) and its time of occurrence (i.e. after dosing).
Hunched posture and/or piloerection was noted in two females at 1000 mg/kg (nos. 79 and 80) on a single occasion. At the low incidence observed, these findings were considered not to be toxicologically relevant.
Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.
One female at 1000 mg/kg (no. 73) was sacrificed at PND 1 due to total litter loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was reduced (statistically significantly) in the first week of the pre-mating period in males and females treated at 1000 mg/kg. Thereafter, growth rate at 1000 mg/kg was comparable to that in controls. Mean body weights at 1000 mg/kg were about 5% lower than those in controls from Day 8 of the pre-mating period (statistical significance was achieved only on Day 8 of the pre-mating period and Day 1 of the lactation period).
Body weights and body weight gain were not affected by treatment at 100 or 300 mg/kg.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption before and after allowance for body weight was decreased in the first week of the pre-mating period in males and females treated at 1000 mg/kg. Food consumption of females at 1000 mg/kg was also lower in the second week of the pre-mating period (before and after allowance for body weight) and during the post-coitum period (mostly before allowance for body weight).
No treatment-related changes in food consumption were noted at 100 or 300 mg/kg.

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

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Mating index
Mating index was not affected by treatment. Except for one control female (no. 46), all females showed evidence of mating.

Fertility and Conception index
Fertility and conception index were not affected by treatment. Except for one control female (no. 41), all mated females were pregnant.

Precoital time
Precoital time was not affected by treatment.

Number of corpora lutea and implantation sites
Numbers of corpora lutea and implantation sites were not affected by treatment.
Compared to the control group, the mean numbers of corpora lutea and implantation sites appeared lower in all groups treated with the test item. The differences were not statistically significant, showed no dose-related trend, and remained within normal limits. Therefore, this finding was considered to be unrelated to treatment.
For females nos. 59 (100 mg/kg) and 63 (300 mg/kg) the number of pups was slightly higher than the number of corpora lutea and/or implantation sites. This was considered to be due to normal resorption of these areas as these enumerations were performed on Day 6 of lactation.

Developmental Data
Gestation index and duration
Gestation index and duration of gestation were not affected by treatment.

Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

One female of the control group (no. 41, mated with male no. 01) was not pregnant, which was considered to be due to the presence of a moderate keratin cyst in her cervix.
Histopathology did not reveal any changes in the reproductive organs that could explain the lack of healthy offspring in two other couples (female/male nos. 46/06 of the control group which had not mated, and nos. 73/33 of the 1000 mg/kg group with total litter loss).
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and spermatogenic staging profiles were normal for all males examined.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
Frequently observed clinical signs included scabs on the snout/head or other parts of the body, wounds at different parts of the body, and blue spot (mostly on the snout). Other clinical signs were noted incidentally (lean appearance, less milk in the stomach, dehydration, deformed front leg(s), swollen hind leg, missing tail tip or toes). The nature and incidence of these findings did not indicate a relation with treatment.
Note: Only days on which clinical signs were present between first and last litter check are presented in the table.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

At first litter check, three litters included one dead pup (litter nos. 43 and 47 of the control group and no. 69 of the 300 mg/kg group). One female at 1000 mg/kg (no. 73) had total litter loss (she delivered only one dead pup). This incidental pup mortality was within normal limits and unrelated to treatment with the test item.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were not affected by treatment.

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings were observed that were considered to be related to treatment.
The nature and incidence of incidental macroscopic findings noted among pups remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Live births
At first litter check, three litters included one dead pup (litter nos. 43 and 47 of the control group and no. 69 of the 300 mg/kg group). One female at 1000 mg/kg (no. 73) had total litter loss (she delivered only one dead pup). This incidental pup mortality was within normal limits and unrelated to treatment with the test item.
The mean number of living pups at first litter check appeared lower in all groups treated with the test item. The differences from the control group were not statistically significant, showed no dose-related trend, and remained well within normal limits. Therefore, this finding was not attributed to treatment.

Viability index
Viability index (number of offspring surviving until planned necropsy as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices across the groups were 96-100%.
During lactation (PND 2 or 3), four pups of the control group (litter nos. 43 and 45) and three pups at 1000 mg/kg (litter nos. 72 and 74) went missing. These missing pups were most likely cannibalized. One pup at 300 mg/kg was found dead at PND 5 (litter no. 62). No toxicological relevance was attributed to these dead/missing pups since the mortality incidence showed no dose-related trend and remained within the range considered normal for pups of this age.

Sex ratio
Sex ratio was not affected by treatment.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

Any other information on results incl. tables

CLINICAL SIGNS SUMMARY

MALES

SIGN (MAX. GRADE) (LOCATION)	WEEK: DAY:	PRE MATING
		1 1	. 2	. 3	. 4	. 5	. 6	. 7	. 1	. 2	. 3	. 4	. 5	. 6	. 7
GROUP 1 (CONTROL)
Skin / fur            Scabs (3)            (Cheek left)            Scabs (3)          (Cheek right)            Scabs (3)            (Tail)	G: % G: % G: %:	. . . . . .	. . . . . .	. . . . . .	. . . . . .	. . . . . .	. . . . . .	. . . . . .	. . . . . .	. . . . . .	1 1 . . . .	1 1 . . . .	1 1 . . . .	1 1 . . . .	1 1 . . . .
GROUP 2 (100 MG/KG)
Secretion / excretion          Salivation (3)	G: %:	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .	. .
GROUP 3 (300 MG/KG)
Secretion / excretion          Salivation (3)	G: %:	. .	. .	1 4	1 A	1 A	1 4	1 4	1 4	1 4	1 4	1 4	1 4	1 4	1 4
GROUP 4 (1000 MG/KG)
Breathing            Rales (3)   Secretion / excretion          Salivation (3)	G: %:   G: %:	. .   . .	. .   . .	. .   1 9	. .   1 9	. .   1 9	1 1   1 9	. .   1 9	. .   1 9	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A

 

SIGN (MAX. GRADE) (LOCATION)	WEEK: DAY:	REPRO PERIOD
		1 1	. 2	. 3	. 4	. 5	. 6	. 7	. 1	. 2	. 3	. 4	. 5	. 6	. 7	. 1	. 2	. 3	. 4	. 5	. 6	. 7
GROUP 1 (CONTROL)
Skin / fur            Scabs (3)            (Cheek left)            Scabs (3)           (Cheek right)            Scabs (3)            (Tail)	G: %: G: %: G: %:	. . 1 1 . .	. . 1 1 . .	. . . . . .	. . . . . .	. . . . . .	. . . . . .	. . . . . .	. . . . . .	. . . . . .	. . . . . .	. . . . . .	. . . . . .	. . . . 1 1	. . . . 1 1	. . . . 1 1
GROUP 2 (100 MG/KG)
Secretion / excretion          Salivation (3)	G: %:	. .	. .	. .	. .	. .	. .	. .	. .	1 1	1 1	1 2	1 2	1 2	1 2	1 2
GROUP 3 (300 MG/KG)
Secretion / excretion          Salivation (3)	G: %:	1 4	1 4	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A	1 A
GROUP 4 (1000 MG/KG)
Breathing            Rales (3)   Secretion / excretion          Salivation (3)	G: % G: %:	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   1 A	. .   2 A	. .   2 A	. .   2 A	. .   1 A	. .   1 A

 

FEMALES

SIGN (MAX. GRADE) (LOCATION)	WEEK: DAY:	PRE MATING
		1 1	. 2	. 3	. 4	. 5	. 6	. 7	. 1	. 2	. 3	. 4	. 5	. 6	. 7
GROUP 1 (CONTROL)
No clinical signs noted
GROUP 2 (100 MG/KG)
No clinical signs noted
GROUP 3 (300 MG/KG)
Secretion / excretion            Salivation (3)	G: %:	. .	. .	1 2	1 A	1 A	1 A	1 A	1 A	1 9	1 9	1 9	1 9	1 9	1 A
GROUP 4 (1000 MG/KG)
Posture          Hunched posture (1)   Skin / fur            Piloerection (1)              Alopecia (3)   Secretion / excretion            Salivation (3)	G: %:   G: %: G: %:   G: %:	. .   . . . .   . .	. .   . . . .   1 2	. .   . . . .   1 9	. .   . . . .   1 9	. .   . . . .   1 9	. .   . . . .   1 9	. .   . . . .   1 9	. .   . . . .   1 9	. .   . . . .   1 9	. .   . . . .   1 9	. .   . . . .   1 9	. .   . . . .   1 9	. .   . . . .   1 A	. .   . . . .   2 A

 

SIGN (MAX. GRADE) (LOCATION)

WEEK: DAY:

REPRO PERIOD

1 1

. 2

. 3

. 4

. 5

. 6

. 7

. 1

. 2

. 3

. 4

. 5

. 6

. 7

. 1

. 2

. 3

. 4

. 5

. 6

. 7

GROUP 1 (CONTROL)

No clinical signs noted

GROUP 2 (100 MG/KG)

No clinical signs noted

GROUP 3 (300 MG/KG)

Secretion / excretion            Salivation (3)

G: %:

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

GROUP 4 (1000 MG/KG)

Posture        Hunched posture (1)   Skin / fur            Piloerection (1)              Alopecia (3)   Secretion / excretion            Salivation (3)

G: %: G: %: G: %: G: %:

. .   . . . .   2 A

. .   . . . .   2 A

. .   . . . .   2 A

. .   . . . .   2 A

. .   . . . .   2 A

. .   . . . .   2 A

. .   . . . .   2 A

. .   . . . .   2 A

. .   . . . .   2 A

. .   . . . .   2 A

. .   . . . .   2 A

. .   . . . .   2 A

. .   . . . .   2 A

. .   . . . .   1 A

. .   . . . .   1 A

. .   . . . .   2 A

1 1   1 2 . .   2 A

. .   . . . .   2 A

. .   . . . .   2 A

. .   . . . .   2 A

. .   . . . .   2 A

 

SIGN (MAX. GRADE) (LOCATION)

WEEK: DAY:

REPRO PERIOD

4 1

. 2

. 3

. 4

. 5

. 6

. 7

. 1

. 2

. 3

. 4

. 5

. 6

. 7

. 1

. 2

. 3

. 4

. 5

. 6

. 7

GROUP 1 (CONTROL)

No clinical signs noted

GROUP 2 (100 MG/KG)

No clinical signs noted

GROUP 3 (300 MG/KG)

Secretion / excretion            Salivation (3)

G: %:

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

1 A

GROUP 4 (1000 MG/KG)

Posture         Hunched posture (1)   Skin / fur            Piloerection (1)              Alopecia (3)   Secretion / excretion            Salivation (3)

G: %: G: %: G: %: G: %:

. .   . . . .   2 A

. .   . . . .   2 A

. .   . . 1 1   2 A

. .   . . 1 1   2 A

. .   . . 1 1   2 A

. .   . . 1 1   2 A

. .   . . 1 1   2 A

. .   . . 1 2   2 A

. .   . . 1 3   2 A

. .   . . 1 3   2 A

. .   . . 1 5   2 A

. .   . . 1 A   2 A

. .   . . 1 A   2 A

. .   . . 1 A   2 A

. .   . . 1 A   2 A

. .   . . 1 A   2 A

. .   . . 1 A   2 A

. .   . . 1 A   2 A

. .   . . 1 A   2 A

. .   . . 1 A   2 A

. .   . . 1 A   2 A

 

G: Median value of the highest individual daily grades

%: Percent of affected animals (0=less than 5%, 1=between 5% and 15%, …., A=more than 95%)

.: Observation performed, sign not present

 

BODY WEIGHTS (GRAM) SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
PRE MATING
DAY 1 WEEK 1	MEAN ST. DEV. N	305 9.9 10	303 6.3 10	305 6.9 10	302 7.1 10
DAY 8 WEEK 2	MEAN ST. DEV N	335 14.0 10	332 10.9 10	335 8.3 10	316 ** 16.4 10
MATING PERIOD
DAY 1 WEEK 1	MEAN ST. DEV. N	357 20.2 10	353 17.1 10	358 9.2 10	338 21.9 10
DAY 8 WEEK 2	MEAN ST. DEV. N	366 21.3 10	368 22.5 10	369 11.4 10	352 27.4 10
DAY 15 WEEK 3	MEAN ST. DEV. N	382 21.3 10	382 26.4 10	379 11.7 10	363 31.1 10

 

FEMALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
PRE MATING
DAY 1 WEEK 1	MEAN ST. DEV. N	207 9.6 10	209 8.0 10	205 8.3 10	206 7.5 10
DAY 8 WEEK 2	MEAN ST. DEV. N	218 8.3 10	218 8.7 10	211 11.6 10	208 * 7.0 10
MATING PERIOD
DAY 1 WEEK 1	MEAN  ST. DEV. N	223 5.6 10	224 10.3 10	221 12.2 10	215 7.0 10
DAY 8 WEEK 2	MEAN ST. DEV. N	252 4.5 4	268 --- 1		236 --- 1
DAY 15 WEEK 3	MEAN ST. DEV. N	269 6.8 3
DAY 22 WEEK 4	MEAN ST. DEV. N	309 54.4 3
DAY 29 WEEK 5	MEAN ST. DEV. N	246 --- 1
DAY 36 WEEK 6	MEAN ST. DEV. N	249 --- 1

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

 

BODY WEIGHTS (GRAM) SUMMARY

FEMALES

F0-GENERATION

		GROUP 1 CONTROL	GROUP 2 100 M/KG	GROUP 3 300 M/KG	GROUP 4 1000 MG/KG
POST COITUM
DAY 0	MEAN ST. DEV. N	227 8.1 6	233 17.5 9	225 10.1 10	230 9.3 9
DAY 4	MEAN ST. DEV. N	241 9.9 6	245 15.6 10	236 12.0 10	230 7.8 10
DAY 7	MEAN ST. DEV. N	249 9.4 6	253 16.4 10	243 10.6 10	238 6.1 10
DAY 11	MEAN ST. DEV. N	264 10.4 6	266 15.2 10	256 11.3 10	253 6.9 10
DAY 14	MEAN ST. DEV. N	276 10.8 6	276 15.9 10	268 12.2 10	263 7.2 10
DAY 17	MEAN ST. DEV. N	298 9.5 6	298 17.5 10	289 14.2 10	281 11.2 10
DAY 20	MEAN ST. DEV. N	335 10.9 6	331 23.9 10	313 27.3 10	311 17.4 10
LACTATION
DAY 1	MEAN ST. DEV. N	257 12.8 8	266 13.4 10	253 11.2 10	243 * 6.3 10
DAY 4	MEAN ST. DEV. N	267 9.2 8	277 15.3 10	261 14.0 10	254 10.4 9

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

 

BODY WEIGHT GAIN (%) SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
PRE MATING
DAY 1 WEEK 1	MEAN ST. DEV. N	0 0.0 10	0 0.0 10	0 0.0 10	0 0.0 10
DAY 8 WEEK 2	MEAN ST. DEV. N	10 1.7 10	10 1.8 10	10 1.5 10	5 ** 3.2 10
MATING PERIOD
DAY 1 WEEK 1	MEAN ST. DEV. N	17 3.5 10	16 3.7 10	17 2.2 10	12 ** 4.7 10
DAY 8 WEEK 2	MEAN ST. DEV. N	20 4.1 10	21 5.4 10	21 2.5 10	16 6.6 10
DAY 15 WEEK 3	MEAN ST. DEV. N	25 4.2 10	26 6.7 10	24 2.5 10	20 7.7 10

 

FEMALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
PRE MATING
DAY 1 WEEK 1	MEAN ST. DEV. N	0 0.0 10	0 0.0 10	0 0.0 10	0 0.0 10
DAY 8 WEEK 2	MEAN ST. DEV. N	6 3.0 10	4 2.6 10	3 2.9 10	1 ** 3.5 10
MATING PERIOD
DAY 1 WEEK 1	MEAN ST. DEV. N	8 4.2 10	8 3.6 10	8 2.6 10	4 3.8 10
DAY 8 WEEK 2	MEAN ST. DEV. N	21 5.3 4	22 -- 1		12 -- 1
DAY 15 WEEK 3	MEAN ST. DEV. N	30 10.6 3
DAY 22 WEEK 4	MEAN ST. DEV. N	50 33.3 3
DAY 29 WEEK 5	MEAN ST. DEV. N	12 -- 1
DAY 36 WEEK 6	MEAN ST. DEV. N	14 -- 1

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

 

BODY WEIGHT GAIN (%) SUMMARY

FEMALES

F0-GENERATION

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
POST COITUM
DAY 0	MEAN ST. DEV. N	0 0.0 6	0 0.0 9	0 0.0 10	0 0.0 9
DAY 4	MEAN ST. DEV. N	6 1.0 6	5 1.9 9	5 2.0 10	5 1.6 10
DAY 7	MEAN ST. DEV. N	10 2.0 6	9 2.2 9	8 2.6 10	9. 2.3 9
DAY 11	MEAN ST. DEV. N	17 1.9 6	15 3.3 9	14 3.5 10	15 2.9 9
DAY 14	MEAN ST. DEV. N	22 2.2 6	19 3.5 9	19 3.9 10	20 3.5 9
DAY 17	MEAN ST. DEV. N	31 3.3 6	29 4.4 9	29 4.6 10	29 4.5 9
DAY 20	MEAN ST. DEV. N	48 5.4 6	44 6.9 9	39 13.0 10	44 6.7 9
LACTATION
DAY 1	MEAN ST. DEV. N	0 0.0 8	0 0.0 10	0 0.0 10	0 0.0 10
DAY 4	MEAN ST. DEV. N	4 3.0 8	4 3.0 10	3 2.2 10	5 2.0 10

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

 

FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
PRE MATING
DAYS 1-8 WEEKS 1-2	MEAN ST. DEV. N (CAGE)	25 0.2 2	25 0.0 2	25 0.1 2	21 0.7 2
DAYS 8-15 WEEKS 2-3	MEAN ST. DEV. N (CAGE)	28 1.2 2	27 0.7 2	28 0.0 2	26 1.7 2
MEAN OF MEANS OVER PRE MATING	MEAN	27	26	27	24
MATING PERIOD
DAYS 1-8 WEEKS 1-2	MEAN ST. DEV. N (CAGE)	29 1.1 2	28 2.3 2	23 4.9 2	24 3.2 2
DAYS 8-15 WEEKS 2-3	MEAN ST. DEV. N (CAGE)	27 1.3 2	26 1.2 2	26 0.9 2	25 0.8 2
MEAN OF MEANS OVER MATING PERIOD	MEAN	28	27	25	25

 

FEMALES

		GROUP 1 CONTROL	GROUP 2 100 M/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
PRE MATING
DAYS 1-8 WEEKS 1-2	MEAN ST. DEV. N (CAGE)	18 0.3 2	17 0.3 2	17 0.3 2	14 0.2 2
DAYS 8-15 WEEKS 2-3	MEAN ST. DEV. N (CAGE)	19 0.3 2	19 1.2 2	19 0.4 2	17 0.1 2
MEAN OF MEANS OVER PRE MATING	MEAN	18	18	18	15

 

FEMALES

F0-GENERATION

		GROUP 1 CONTROL	GROUP 2 100 M/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
POST COITUM
DAYS 0-4	MEAN ST. DEV. N	21 4.7 6	19 1.9 9	19 1.8 10	17 * 1.4 9
DAYS 4-7	MEAN ST. DEV. N	23 3.9 6	21 2.1 10	21 2.2 10	19 ** 1.2 10
DAYS 7-11	MEAN ST. DEV. N	23 1.4 6	22 1.8 10	22 2.2 10	20 * 1.5 10
DAYS 11-14	MEAN ST. DEV. N	23 2.2 6	22 2.4 10	22 1.8 10	20 ** 1.0 10
DAYS 14-17	MEAN ST. DEV. N	23 2.9 6	24 2.2 10	23 1.6 10	22 1.8 10
DAYS 17-20	MEAN ST. DEV. N	25 2.4 6	25 2.1 10	24 2.2 10	22 * 1.6 10
MEAN OF MEANS		23	22	22	20
LACTATION
DAYS 1-4	MEAN ST. DEV. N	26 2.9 8	27 6.3 10	25 5.6 10	27 3.8 9

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

 

MACROSCOPIC FINDINGS SUMMARY

MALES

	GROUP 1 CONTROL	GROUP 2 100 M/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
END OF TREATMENT
Animals examined Animals without findings	10 8	10 8	10 9	10 8
Animal affected	2	2	1	2
Epididymides            Nodule(s) Cowper’s gland            Agenesis Thymus            Focus/foci Skin            Scab formation	0   0   1   1	0   1   1   0	0   1   0   0	1   0   1   0

FEMALES

	GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
INTERCURRENT DEATH
Animals examined Animal affected				1 1
General observations            Total litter loss				1
END OF TREATMENT
Animal examined Animals without findings	10 8	10 9	10 10	9 8
Animals affected	2	1	0	1
Clitoral glands            Focus/foci Skin            Alopecia	2   0	1   0	0   0	0   1

 

ORGAN WEIGHTS (GRAM) SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
END OF TREATMENT
BODY W. (GRAM)	MEAN ST. DEV. N	381 23 10	382 28 10	381 11 10	363 32 10
TESTES (GRAM)	MEAN ST. DEV. N	3.57 0.25 10	3.43 0.22 10	3.56 0.27 10	3.60 0.29 10
EPIDIDYMIDES (GRAM)	MEAN ST. DEV. N	1.092 0.079 10	1.101 0.077 10	1.142 0.074 10	1.094 0.068 10

 

REPRODUCTION DATA SUMMARY

	GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
Females paired Females mated Pregnant females Females with total litter loss Females with living pups on Day 1	10 9 8 0 8	10 10 10 0 10	10 10 10 0 10	10 10 10 1 9
Mating index (%) (Females mated/Females paired) * 100 Fertility index (%) (Pregnant females/Females paired) * 100 Conception index (%) (Pregnant females/Females mated) * 100 Gestation index (%) (Females with living pups on Day 1/Pregnant females) * 100	90   80   89   100	100   100   100   100	100   100   100   100	100   100   100   90

 

PRECOITAL TIME

F0-GENERATION – POST COITUM

DAY OF THE PAIRING PERIOD	GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
NUMBER OF FEMALES MATED
1 2 3 4 5 13 14	1 3 3 1 1 - -	3 1 1 4 - 1 -	4 1 2 3 - - -	6 1 1 1 - - 1
MEDIAN PRECOITAL TIME MEAN PRECOITAL TIME N	3 2.8 9	4 3.7 10	3 2.4 10	1 2.9 10

 

CORPORA LUTEA IMPLANTATION SITES SUMMARY

FEMALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
NECROPSY
Corpora Lutea	MEAN ST. DEV. N	15.1 3.0 8	12.6 4.3 10	13.4 2.2 10	12.8 1.9 10
Implantations	MEAN ST. DEV. N	13.0 2.6 8	11.2 4.2 10	12.1 2.1 10	10.9 3.1 10

 

DEVELOPMENTAL DATA

F0-GENERATION – LACTATION

	GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 M/KG	GROUP 4 1000 MG/KG
LITTERS            TOTAL	8	10	10	10
DURATION OF GESTATION            MEAN (+)            ST. DEV.            N	21.4 0.5 8	21.4 0.5 10	21.1 1.2 10	21.4 0.5 10
DEAD PUPS AT FIRST LITTER CHECK            LITTERS AFFECTED (#)            TOTAL            MEAN (+)            ST. DEV.            N	2 2 0.3 0.5 8	0 0 0.0 0.0 10	1 1 0.1 0.3 10	1 1 0.1 0.3 10
LIVING PUPS AT FIRST LITTER CHECK            % OF MALES / FEMALES (#)            TOTAL            MEAN (+)            ST. DEV.            N	55 / 45 97 12.1 2.9 8	50 / 50 105 10.5 4.2 10	48 / 52 95 9.5 3.4 10	48 / 52 94 9.4 3.8 10
POSTNATAL LOSS            % OF LIVING PUPS            LITTERS AFFECTED (#)            TOTAL (#)            MEAN (+)            ST. DEV.            N	4.1 2 4 0.5 1.1 8	0.0 0 0 0.0 0.0 10	1.1 1 1 0.1 0.3 10	3.2 2 3 0.3 0.7 10
VIABILITY INDEX (#)	95.9	100.0	98.9	96.8

Viability index = (Number of alive pups before planned necropsy/Number of pups born alive) * 100

+/++ Steel-test significant at 5% (+) or 1% (++) level

#/## Fisher’s Exact test significant at 5% (#) or 1% (##) level

 

BODY WEIGHTS OF PUPS (GRAM)

F0-GENERATION – LACTATION

DAY	SEX		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
1	M	MEAN ST. DEV. N	6.1 0.6 8	6.5 0.7 10	6.5 0.8 9	6.3 0.8 9
	F	MEAN ST. DEV. N	5.9 0.7 8	6.0 0.7 10	6.3 0.7 10	6.0 0.6 10
	M+F	MEAN ST. DEV. N	6.0 0.6 8	6.2 0.7 10	6.4 0.7 10	6.1 0.7 10
4	M	MEAN ST. DEV. N	9.1 1.2 8	9.4 1.6 10	9.6 1.4 9	9.5 1.3 9
	F	MEAN ST. DEV. N	8.7 1.3 8	8.5 1.8 10	9.4 1.2 10	9.2 1.3 9
	M+F	MEAN ST. DEV. N	9.0 1.2 8	9.0 1.7 10	9.6 1.3 10	9.3 1.3 9

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

Applicant's summary and conclusion

Conclusions:

Based on the results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.

Executive summary:

Title

Reproduction/developmental toxicity screening test of MLA-3202 in rats by oral gavage.

 

Guidelines

The study was based on the following guidelines:

-OECD 421, Reproduction/Developmental Toxicity Screening Test, July 1995

-OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

 

Rationale for dose levels

Dose levels for this reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg, based on preliminary results of a 28-day repeated dose toxicity study (Test Facility Project 514867).

 

Study outline

The test item, formulated in water, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females that delivered were treated for 42-46 days (most females) or 56 days (two females), i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 5-6 days of lactation. Female nos. 41, 46 and 73 which failed to deliver healthy offspring were treated for 44, 53 and 39 days, respectively.

 

Evaluated parameters

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), body weight and food consumption (at least at weekly intervals), macroscopy at termination, organ weights (testes, epididymides) and histopathology on reproductive organs. In addition, the following reproduction/developmental parameters were determined: mating, fertility and conception indices, precoital time, numbers of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, macroscopy). Formulations were analyzed once during the study to assess accuracy and homogeneity.

 

Results/discussion

Accuracy and homogeneity of formulations were demonstrated by analyses.

 

Parental results:

No parental toxicity was observed up to the highest dose level tested (1000 mg/kg).

The salivation noted after dosing in all rats treated at 300 or 1000 mg/kg and in a few males at 100 mg/kg was considered to be a physiological response rather than a sign of systemic toxicity.

Body weight gain and food consumption were reduced at 1000 mg/kg during the first week of the treatment period in both sexes. Food consumption of females at 1000 mg/kg was also slightly lower in the second week of the pre-mating period and during the post-coitum period.

Body weights were reduced to only a limited degree (about 5%). These findings were considered not to be of toxicological relevance.

 

Reproductive and developmental results:

No reproduction or developmental toxicity was observed up to the highest dose level tested (1000 mg/kg).

 

Conclusion

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.