Dimethyl (p-methoxybenzylidene)malonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A NOAEL of 1000 mg/kg bw/day for reproductive and developmental effects could be derived from a reliable combined repeated

dose and reproduction screening study in rats.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Justification for study design:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provides initial information on possible effects on male and female reproduction performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats - HsdHan: WIST rats; Conventionally bred (In-house random bred)

Sex:

male/female

Details on test animals or test system and environmental conditions:

No. of groups: 6
Main groups:
Vehicle control (G1)
Low dose (G2)
Mid dose (G3)
High dose (G4)

Recovery Groups:
Vehicle control recovery (G1R)
High dose recovery (G4R)

No. of animals for acclimatization: 60 males and 60 females

No. of rats/group: Main groups: 10 males + 10 females per group; Recovery groups: 5 males + 5 females per group; Total = 100 (50 males + 50 females)

Age at treatment: 16-17 weeks

Body weight range at the start of treatment: Males: 370.61 to 478.55 g; Females: 235.23 to 310.02 g

Route of administration:

oral: gavage

Vehicle:

other: 1 % (w/v) Na CMC (medium viscosity) in Milli-Q water

Details on exposure:

TREATMENT:
Males: The dose formulation was administered to the rats of the specific groups once daily at approximately the same time each day (varying by +/- 3 hours) for 2 weeks prior to mating and the treatment was continued during the mating period and approximately two weeks post mating.
Females: The dose formulations were administered to the specific group of rats once daily at approximately the same time each day (varying by +/- 3 hours) throughout the treatment period. Treatment was done 2 weeks prior to the mating period and continued through mating, pregnancy and up to lactation day 13, after which, pups were sacrificed on lactation day 13 and parental females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed).
The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by +/- 3 hours).
The volume of dose administered was 10 mL/kg Bwt throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at 10 mL/kg Bwt.
The vehicle and the test item was not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days following the treatment period.
Homogeneity of the dose formulations during sampling/gavaging was maintained by constant stirring using a magnetic stirrer.

Details on mating procedure:

One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until there is evidence of sperms in the vaginal smear and/or vaginar plug or for a maximum period of 2 weeks. Subsequently, pregnant females were housed individually until lactation day 14. The females not mated within 14 days of pairing with the first male was placed with a second proven male of the same group and the presence of sperm in the vaginal smear and/or vaginal plug is not confirmed within 7 days, that female was sacrificed 24-26 days after last day of mating period. All the mated females were maintained till they litter or for a maximum period of at least 25 days from the last day of cohabitation. Not littered females were sacrificed after 25 days from the day they are found of sperm positive (by vaginal smear examination).
The day of confirmed mating was designated as Gestation Day 0 (GD 0). The pre-coital time was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

DOSE FORMULATION PREPARATION:
The dose formulations were prepared daily prior to start of the treatment and prepared on need basis and used within the stability period.
Following procedure was followed when 150 mL of dose formulation prepared:
The quantities of 1500 (G2), 4500 (G3) and 15000 (G4) mg of test item was weighed in a glass beaker and transferred to the mortar. The test item was ground to a fine powder in the mortar using pestle. Small volume of vehicle (1 % (w/v) NaCMC in Milli-Q water) was added in a stepwise manner to mortar with continuous trituration till a uniform suspension was obtained. The micture was transferred into the measuring cylinder. Further, a snall volume of vehicle was added to the mortar and rinse with vehicle to ensure the test item was completely transferred. The final volume was made up with vehicle and to attain a desired concentration.

DOSE FORMULATION ANALYSIS:
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and once during Week 4 of the treatment and were sent to Analytical R&D of Advinus Therapuetics Limited. For each set, duplicate samples were drawn from top, middle and bottom layers of each preparation and in case of control, duplicate samples from only middle layer was drawn.
The analysis was done as per the method validated under Advinus Study No. G9993. One set of samples were analyzed for concentration analysis and other set (second set) of samples were stored at ambient conditions for reanalysis purpose as a backup and the second set of samples were discarded, as the analysis results of first set of samples were within the limits.

Duration of treatment / exposure:

see 'Details on exposure'

Frequency of treatment:

see 'Details on Exposure'

Details on study schedule:

see 'Details on Exposure'

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control group (G1)

Dose / conc.:

100 mg/kg bw/day

Remarks:

Low dose group (G2)

Dose / conc.:

300 mg/kg bw/day

Remarks:

Mid dose group (G3)

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose group (G4)

No. of animals per sex per dose:

10 males + 10 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

SELECTION OF DOSE LEVELS AND DOSE JUSTIFICATION
Three dose levels of 100 (low dose), 300 (mid dose) and 1000 (high dose) mg/kg bw/day were selected for groups G2, G3 and G4 respectively as recommended by the sponsor.
In addition to the test doses, vehicle control and vehicle control recovery groups were included. Animals in the vehicle control and recovery were handled in a manner similar to the treatment groups except for test item administration.

Parental animals: Observations and examinations:

See in detail under "Any other information on materials and methods...", below

Oestrous cyclicity (parental animals):

Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select for the study females with regular 4-5 days cyclicity. Vaginal smears were also monitored daily from the beginning of the treatment period until evidence of mating. The time interval (in days) from the diestrus of an oestrus cycle to the next diestrus was considered as the oestrus cycle length of an animal. The overall pattern of each female was characterized as regularly cycling (having recurring 4-5 day cycles).
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Litter observations:

a. At birth, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded.
b. The number of pups born (litter size), sex and individual pup body weight of male and female pups on Days 0, 4 and 13 post-partum were recorded.
c. On Day 4, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Blood samples was collected from two of the surplus pups, pooled, and used for determination of serum T4 levels. If there were insufficient pups in a litter to have two surplus pups, two of the pups that were typically retained were used for blood collection for serum T4 assessments. When litter size dropped below the culling target (8 pups/litter), preference was given to remove two female pups for PND 4 blood samples in order to retain more male pups for observation of nipple retention on post-natal day (PND) 13.
d. After standardization, the individual pup body weight was recorded on Day 13 of lactation.
e. The ano-genital distance (AGD) of each pup was measured on postnatal day (PND) 0 and pup body weight was recorded.
f. All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.
g. Fertility index for dams, sires as well as the pup survival index till LD 4 was calculated.

Postmortem examinations (parental animals):

All adult animals and pups were examined macroscopically for any structural abnormalities and pathological changes. The adult animals sacrificed at term were fasted overnight (water allowed), exsanguinated under isoflurane anaesthesia and weighed. The rats were subjected for detailed necropsy by a veterinary pathologist and findings were recorded. All the surviving pups were necropsied on LD 13 and findings were recorded. Dead pups were examined for possible defects and/or cause of death. The number of implantation sites was recorded for all the dams.

Statistics:

The statistical analysis of the experimental data was carried out using the validated package in excel and also using licensed copies of SYSTAT Statistical package ver. 12.0. All quantitative variables like body weight, food intake, terminal body weight, clinical pathology (haematology clinical chemistry and coagulation) parameters, thyroid hormone levels, organ weights and organ weight ratios were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene's test) within the group before performing a one-factor analysis of variance (ANOVA) modeling by treatment groups. Non-optimal (non-normal or heteroscedastic) data were transformed, before using ANOVA. Means between treatment groups and vehicle control group were compared using Dunnett's test.
Pre-implantation loss (%), post-implantation loss (%), no. of corpora lutea, implantations, pre-coital interval, sex ratio (%) and gestation length (days) were analysed after suitable transformation of the data. One-way ANOVA was carried out for the transformed data. Dunnett's pair-wise comparison of the treated means with the control mean was done for the significant group differences.
Z test was performed for testing the differences in proportions for mating and fertility indices.
Statistically significant differences (p <= 0.05), indicated by the aforementioned tests was designated throughout the report as stated below:
+/- : Significantly higher (+) / lower (-) than the vehicle control group
a+/a- : Significantly higher (a+) / lower (a-) than the vehicle control recovery group

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed at any of the doses tested. However, clinical sign of sparse hair loss at right and left flank was observed in a female (Rt1396) of control group and one male rat (Rt1473) of high dose recovery group was considered spontaneous finding and not related to treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed at any of the doses tested.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males, body weights: No treatment related variations in the mean body weights at all the dose groups during the treatment period. The mean body weight was significantly lower in high dose recovery animals.
Females, body weights: No treatment related variations in the mean body weights at all the dose groups either during the treatment period or recovery periods.

Maternal Body Weights and Food Intake during the Gestation Period: No significant changes in maternal body weight and food consumption were observed at any of the doses tested whn compared to the vehicle control group.

Maternal Body Weights and Food Intake during the Lactation Period: No significant changes in maternal body weight and food consumption were observed at any of the doses tested when compared to the vehicle control group. However, at 1000 mg/kg bw/day, the body weight change was significantly higher during the Days 0-13 and considered incidential as there were no change in the mean body weight.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: The Food consumption was unaffected by the treatment at 100 mg/kg bw/day dose groups. At 300 mg/kg bw/day, the food consumption was significantly lower on week 6 and was considered incidential because of isolated occurrence and there was no associated change in the body weight. At 1000 mg/kg bw/day, the food consumption was significantly lower and considered treatment related finding. In the 1000 mg/kg bw/day dose recovery group, the food consumption was also significantly lower on weeks 2 and 3 and hence were considered to be transient and not of biological significance.

Females: The food consumption was unaffected by the treatment at 100, 300 and 1000 mg/kg bw/day dose groups. In the high dose recovery group, the food consumption was significantly lower on weeks 1 and 3 and was considered incidential because of isolated occurrence and there was no associated change in the body weight.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Males & Females - Home cage, Handling, Open field and Sensory observations: No treatment-related abnormalities were observed in all the tested dose groups either during treatment period or recovery periods.

Description (incidence and severity):

MALES:
-Neuromuscular observations: No treatment-related variations in the hind limb footsplay and fore limbs and hind limbs grip strength either during the treatment period or recovery periods.
-Motor activity: The following statistically significant variations were observed in the motor activity of rats when compared to respective vehicle control group:
Lower: Stereotypic time at interval 1, interval 3 and total at the mid dose; Ambulatory time at interval 1 at the high dose recovery.
The above observed statistical variations in the motor activity measurement were considered to be incidental as the observed changes did not follow the dose proportion and there were no changes in the home cage of open field observations.

FEMALES:
- Neuromuscular observations: No treatment-related variations in the hind limb footsplay and fore limbs and hind limbs grip strength either during the treatment period or recovery periods. However, higher hindlimbs footsplay value was observed at mid dose group and was considered incidential because of the lack of any dose relationship.
- Motor activity: No treatment-related changes were observed in the motor activity at all the tested doses.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cyclicity was evaluated for its length and normality by examining the vaginal smears daily for two weeks during treatment period (prior to cohabitation). The calculated mean oestrous cycle length was 4.49, 4.58, 4.57 and 4.79 days in vehicle control, 100, 300 and 1000 mg/kg bw/day doses, respectively. The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.

Reproductive performance:

no effects observed

Description (incidence and severity):

Fertility index:
No treatment-related effects were observed with respect to mean pre-coital time, gestation length and fertility indices when compared to control means.
the significantly lower male mating and fertility indices at all the doses tested were observed when compared to vehicle control group. These changes were considered incidential, as there changes were marginal and within the in-house historical control data range (Male mating index: 90-100 %; Male fertility index: 80 - 100 %).
The significantly lower female fertility index at 100 and 1000 mg/kg bw/day doses was considered toxicologically insignificant due to lack of dose relationship.

Uterine/Implantation Data:
No treatment-related effects were observed with respect to corpora lutea, implantations, and percentage of pre and post implantation losses when compared to control means.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

haematology

clinical biochemistry

urinalysis

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: NOAEL for reproductive performance

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

Remarks on result:

other: NOAEL for systemic toxicity

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Survival Data of Pups:
Test item had no treatment-related effects on the number of pregnancies, number littered, mean litter size, and number of dead pups at first observation. There were no external abnormalities in live or dead pups in any of the groups.
No treatment-related changes were observed in the survival data of pups up to LD 4 at 100 and 300 mg/kg bw/day. However, lower survival data of pups observed on Day 4 at 1000 mg/kg bw/day which was considered to be due to dams Rt1456, where partial pups were dead and Rt1457, where all pups were dead and cannibalized during LDs 1-2. These isolated changes that can be observed spontaneously in rats of this strain and were considered to be incidential.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Number and Body Weight of Pups during the Lactation Period:
The mean number of male, female and total pups per litter were unaffected by the treatment at 100, 300 and 1000 mg/kg bw/day dose groups. However, at 1000 mg/kg bw/day, the mean number of total pups per litter was significantly lower on lactation Days 4 and 13.
The mean weight of male, female and total pups per litter were unaffected by the treatment at 100, 300 and 1000 mg/kg bw/day dose groups. However, at 100 mg/kg bw/day, the mean weight of total pups per litter were significantly higher on lactation Day 13 and considered to be incidential because of the lack of dose relationship.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Ano-genital Distance (AGD):
No changes attributable to test item were detected in the AGD, body weight and ratio of AGD to the cube root of body weight of either sex.
However, the body weight of male pups was significantly lower at 300 mg/kg bw/day when compared to control group and considered to be incidential because of the lack of dose relationship.
The body weights of female pups were significantly lower at 300 and 1000 mg/kg bw/day when compared to control group.

Areolae/Nipple retention in Pups:
The male pups did not exhibit areola/nipple retention on PND 13.

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Survival Data of Pups:
Test item had no treatment-related effects on the number of pregnancies, number littered, mean litter size, and number of dead pups at first observation. There were no external abnormalities in live or dead pups in any of the groups.
No treatment-related changes were observed in the survival data of pups up to LD 4 at 100 and 300 mg/kg bw/day. The mean number of male, female and total pups per litter were unaffected by the treatment at 100, 300 and 1000 mg/kg bw/day dose groups. However, at 1000 mg/kg bw/day, the mean number of total pups per litter was significantly lower on lactation Days 4 and 13. However, lower survival data of pups observed on Day 4 at 1000 mg/kg bw/day which was considered to be due to dams Rt1456, where partial pups were dead and Rt1457, where all pups were dead and cannibalized during LDs 1-2. These isolated changes that can be observed spontaneously in rats of this strain and were considered to be incidential.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight of male pups on lactation day 0 was significantly lower at 300 mg/kg bw/day when compared to control group and considered to be incidential because of the lack of dose relationship.
The body weights of female pups on lactation day 0 were significantly lower at 300 and 1000 mg/kg bw/day when compared to control group, but not clearly dose-related. However, this effect was compensated during lactation and the body weights were no longer significantyl altered at end of lactation:
The mean weight of male, female and total pups per litter were unaffected by the treatment at 100, 300 and 1000 mg/kg bw/day dose groups. However, at 100 mg/kg bw/day, the mean weight of total pups per litter were significantly higher on lactation Day 13 and considered to be incidential because of the lack of dose relationship.

Other effects:

no effects observed

Description (incidence and severity):

Ano-genital Distance (AGD):
No changes attributable to test item were detected in the AGD, body weight (see above) and ratio of AGD to the cube root of body weight of either sex.
Areolae/Nipple retention in Pups:
The male pups did not exhibit areola/nipple retention on PND 13.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

other: Anogenital distance and nipple retention

Remarks on result:

other: NOAEL for developmental effects

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

no

Conclusions:

- Daily oral (gavage) administration of the test item to male and female Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day for 2 weeks prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) did not induce any adverse effects on fertility and reproductive perfomance. The No Observed Adverse Effect Level (NOAEL) of the test item is considered to be 1000 mg/kg bw/day.

-Considering the changes observed in the body weights and food consumption, prostate and seminal vesicle with coagulating gland weights, increased kidney weight and grossly white foci in nonglandular mucosa of stomach in male rats, microscopic changes in stomach (hyperkeratosis and epithelial hyperplasia in nonglandular mucosa and mucosal necrosis) and kidneys (dilated tubules and tubular degeneration/regeneration) in both sexes at 1000 mg/kg bw/day, the no observed adverse effect level (NOAEL)" for systemic toxicity is considered to be 300 mg/kg bw/day.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study was provided initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

The test item was suspended in vehicle [1 % (w/v) Carboxymethyl cellulose (CMC) sodium salt in Milli-Q water] and administered by oral gavage at the dose levels of 100, 300 and 1000 mg/kg bw/day to low (G2), mid (G3) and high dose (G4)/ high dose recovery (G4R) group rats, respectively. A concurrent control (G1) and a control recovery group (G1R) of rats received vehicle alone. The dose volume administered was 10 mL/kg bw/day. The main groups i.e., G1 to G4 consisted of 10 male and 10 female rats per group and recovery groups consisted of 5 male and 5 female rats per group. The prepared dose formulations were administered once daily to specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to lactation day 13 (for females). In the control and high dose recovery groups, the treatment period was followed by a 14 day no treatment (recovery) period. The recovery period of the study was started from the first scheduled kill of dams.

The identity of the test item was provided by the study Sponsor by a Certificate of Analysis (CoA). The stability and homogeneity of test item in the vehicle was carried out under Advinus Study No. G9993. Based on the results, the test item was found to be stable for up to 9 days at 1 and 100 mg/mL concentrations when stored at room temperature and refrigeration condition. The dose formulations were analysed for active ingredient (a. i.) concentration on Day 1 and during Week 4 of the treatment period. The results indicated that the analysed concentrations were within +/- 15 % of variations from the nominal concentrations.

All rats were observed for clinical signs once daily. Body weight was recorded at the beginning of the treatment, weekly thereafter and at termination. Food consumption was recorded weekly except during the cohabitation period. Female rats were also weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4, 7, 11 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on LD 0. All the survived male pups were exampined for the appearance nipples/aerolae on PND 13. The functional observation battery was done shortly before sacrifice for randomly selected 5 males and 5 females from each group. For recovery groups, functional observation battery was done prior to sacrifice. Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed in randomly selected 5 males and 5 females from each group at the end of the pre-mating period for main groups and at the end of recovery period from all animals of recovery groups. T4 and TSH hormone analysis was performed in all males at termination, all dams at termination on LD14 and available two pups per litter on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected. Tissues collected from randomly selected 5 males and 5 females in the control and high dose groups were examined microscopically for histopathological changes. A gross necropsy was performed on all dams on LD 14 and study plan specified tissues were collected. All the surviving pups were sacrificed on LD 13 and thyroid gland from available one male and one female pup from each litter was collected for histopathological examination. The reproductive organs of infertile males (Rt1404, Rt1429 and Rt1443) and females (Rt1419 and Rt1460) rats were examined microscopically.

Under the experimental conditions employed, the following results were obtained:

- There were no treatment-related clinical signs or mortality observed at any of the doses tested.

- No treatment-related neurological abnormalities/dysfunctions were observed at all the doses tested.

- At 1000 mg/kg bw/day, the mean body weights and food consumption were significantly lower in males when compared to vehicle control group.

- The body weights and food consumption were unaffected by the treatment during gestation and lactation period at all the doses tested.

- Treatment had no effect on pre-coital time or gestation length, oestrous cycle length, mating and fertility parameters in both sexes.

- There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups. The survival index was not altered by the treatment at any of the doses tested.

- No treatment-related changes in the ano-genital distance and ano-genital index were observed at any of the doses tested when compared to the control group.

- The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

- The test item administration did not reveal any treatment related changes in the hematology, coagulation and clinical chemistry parameters of both males and females.

- At 1000 mg/kg bw/day, the terminal fasting body weight was decreased when compared to the control group.

- At 1000 mg/kg bw/day, test item-related changes includes decrease in weights of prostate and seminal vesicle with coagulating gland of males, inreased weights of ovaries with oviducts and uterus with cervix in female rats. Increased absolute and relative kidney weight in male rats were observed.

- There were no significant changes in thyroid stimulating hormone (TSH) and thyroxin hormone levels in adult animals and pups. there were no test item-related changes in thyroid weights of pups. There were no test item-related microscopic changes in thyroid gland of pups.

- Test item-related histopathological changes were observed in stomach (hyperkeratosis and epithelial hyperplasia in nonglandular mucosa; and mucosal necrosis) and kidneys (dilated tubules and tubular degeneration/regeneration) in both sexes at 1000 mg/kg bw/day, This change showed complete recovery at the end of recovery period. There were no test item-related changes in male and female reproductive organs.

- Reduction observed in absolute and relative weights of prostate and seminal vesicle with coagulating gland of males at 1000 mg/kg bw/day without any associated microscopic changes was attributed to reduced body weight observed in these rats and considered as a test item-related secondary change.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable study, GLP-compliant and according to OECD guideline 422

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Quality of whole database:

Developmental effects were examined in the OECD 422 study (see above) and no effects were observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action possibly observed effects would be regarded as relevant for humans.

Justification for classification or non-classification

As no reproductive or developmental effects were oberved up to the highest dose (1000 mg/kg bw/day), no classification is required according to Regulation (EC) No. 1272/2008 (CLP).