Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The results of a 2-generation-study with a structurally related compound in rats by the oral route gave no indication of a fertility impairing effect. The NOEL was determined to be 21 - 26 mg/kg bw/day based on dosage and effects during lactation.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Jan. 22, 1988 to Oct. 16, 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Estrous cycle not measured prior to mating, sperm parameters not examined. Content in feed not adjusted for food consumption
Qualifier:
according to
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmBH
- Age at study initiation: aproximately 6 weeks
- Weight at study initiation: (P) Males: 165-220g; Females: 140-175g;
- Fasting period before study: None
- Housing:individually in solid floor macrolone cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: February 1, 1988 To: February 1, 1989
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): every 2 weeks
- Mixing appropriate amounts with (Type of food): powdered food
- Storage temperature of food: room temp

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not applicable, test substance mixed in diet
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 3 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
- Each male was mated with one female from the same dose groups for up to 3 weeks
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged: individually except during lactation, when each female was housed with its litter.
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
10 gram samples of the test article were taken once after receipt of the test article (before initiation of the study), once after the start of the in-life phase, once before start of treatment of the Fl generation, and once at the end of the in-life phase and sent to the study sponsor on 26.05.1987, 21.01.1988, 30.06.1988, or 03.02.1989, respectively, for analysis.
In order to verify accurate preparation of the admixtures of the test article to the powdered diet, formulations prepared for this study were analysed for determination of test article concentration, homogeneity, and stability (over two and four week intervals). Analytical examinations of
the formulation preparations from 29.01.1988 until 20.06.1988 (prior to the completion of the new analytical laboratory at Hazleton Laboratories Deutschland GmbH) were performed by Mr. Frank Harhoff, Institut fiir Laboratoriumsmedizin, BrauhausstraBe 4, 4600 Dortmund 1, West Germany. An HPLC method was used.
Duration of treatment / exposure:
For both P and F1 generations (during first pre-mating treatment of 100 days, during mating aprox 21 days, gestation approx 21 days and lactation approx 21 days;second pre-mating treatment of 14 days, during second mating aprox 21 days, gestation approx 21 days and lactation approx 21 days ).
Frequency of treatment:
Daily - In diet ad libitum
Details on study schedule:
After 100 days of premating treatment, the P parental animals were mated for up to 21 days and the females were allowed to litter and to rear their offspring (F1a generation) to weaning. Approximately 14 days after all the F1a offspring were weaned, the P parental animals were paired again, within the dose groups, for up to 21 days. Alternative pairings from the first mating were employed. The P females were allowed to litter and to rear their offspring (F1b generation) to weaning.
After a 100 days maturation period after weaning, the F1 parental animals (selected from the F1a offspring) were mated for up to 21 days and the females were allowed to litter and to rear their offspring (F2a generation) to weaning. Approximately 14 days after all the F2a offspring were weaned, the F1 parental animals were paired again, within the dose groups, for up to 21 days. Alternative pairings from the first mating were employed. The F1 females were allowed to litter and to rear their offspring (F2b generation) to weaning.
Dose / conc.:
300 ppm
Remarks:
corresponding to 21 - 26 mg/kg bw/day; peak exposure of up to 53 mg/kg bw/day during lactation
Dose / conc.:
900 ppm
Remarks:
corresponding to 60 - 80 mg/kg bw; peak exposure of up to 124 mg/kg bw/day during lactation
Dose / conc.:
1 800 ppm
Remarks:
corresponding to 120 - 160 mg/kg bw/d; peak exposure of up to 246 mg/kg bw/day during lactation
No. of animals per sex per dose:
P generation = 30/sex/group
F1 generation = 25/sex/group (pairing 1), 26males and 24 fermales/group (pairing 2)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: the dose levels were selected on the basis of the results from previous acute oral, 90 day oral, oral teratogenicity, and dose range-finding studies.
- Rationale for animal assignment (if not random): random

Administration at 300 ppm corresponds approximately to a 21 to 26 mg/kg/day dose level if calculated as the overall mean of the weekly mean values of mean daily test article intake (the overall mean of P and F1 males corresponds to approximately
21 mg/kg/day dose level - range of the weekly mean values 14.6 to 47.3 mg/kg/day - and the overall mean of P and F1 females corresponds to
approximately 26 mg/kg/day dose level if the gestation and lactation periods are excluded - range of the weekly mean values 20.5 to 47.6 mg/kg/day).
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: NA
Oestrous cyclicity (parental animals):
Not reported.
Cervix, ovaries, uterus, and vagina tissues were examined by the study histopathologist
Sperm parameters (parental animals):
Parameters examined in all male parental generations: epididymides, prostate, seminal vesicles, and testes tissue were examined by the study histopathologist
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, physical developement

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals shortly after their second mating period.
- Maternal animals: All surviving animals after weaning of the second litter.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations in F1 generation.

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissues were prepared for microscopic examination and weighed.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed shortly after weaning or after selection is completed (age at selection not clear).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
Tissues were prepared for microscopic examination and weighed.
Statistics:
Analysis of Variance followed by the Student-Newman-Keuls test for multiple group comparisons.
Reproductive indices:
Mating performance (d)
Insemination index (%)
Fecundity index
Fertility index
Gestation index
Offspring viability indices:
Live birth index (%)
Weaning index (%)
Viability index (d4-1)
Viability index (d7-4)
Viability index (d7-14)
Viability index (d14-21)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Swelling of mouth and forelimb, loss of fur, blood in bedding, inflammation of eyes, cystoliths in urinary bladder, and atrophy of testes were observed in a few animals of varying dose groups; however, due to their nature and low incidence, these clinical observations and necropsy findings were considered incidental.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: mean body weight gain of high dose group was slightly lower than control group
Females: mean body weight gain of high dose group was slightly lower than control group and statistically significant weeks 2 and 7 of treatment and day 14 to 21 of lactation
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1800 ppm (group 4), mean daily food consumption of the male animals of the P generation was slightly reduced prior first and second mating. This is considered attributable to treatment. In the females of the P generation, mean daily food consumption of the animals of group 4 (1800 ppm) was reduced prior first mating and continuously until second lactation.
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):
A few microscopic changes were observed however they were considered to be incidental findings or background pathology
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: During first lactation, mean daily food consumption was dosage-relatedly reduced in groups 3 (900 ppm) and 4 (1800 ppm) and comparable with the control in group 2 (300 ppm). Differences between groups 3 (300 ppm) and 4 (1800 ppm) and the control group wer
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Indices did not reveal any differences between dose groups and control.
Dose descriptor:
NOEL
Effect level:
300 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the male animals, mean body weight gain prior and during first and second mating was - if compared for each of the individual intervals recorded - generally comparable in all groups. For only few of these individual intervals prior and during first mating, the observed differences between groups 3 (900 ppm) or 4 (1800 ppm) and the control were statistically significant; in most of these cases, body weight gain was significantly lower than the control. If the overall treatment period prior start of first mating is taken as the basis for comparison, mean body weight gain of all dose groups was lower than in the control group. The difference to the control group was biggest in group 4 (1800 ppm). This finding was therefore considered to be treatment-related in the high dose group. Body weight development in females was not different to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean daily food consumption of the male animals of group 4 (1800 ppm) was lower than in the control group. The female animals in group 4 (1800 ppm) of this generation showed reduced mean daily food consumption during first lactation, prior second mating, and during second lactation, or in group 3 (900 ppm), mainly prior second mating.
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:
effects observed, non-treatment-related
Description (incidence and severity):
In the male animals of the Fl generation, the lower mean spleen weight and the moderately increased mean kidney weight in group 4 (1800 ppm) as well as the slightly increased mean kidney weight in group 3 (900 ppm) cannot definitively be related to treatment but a treatment-relationship is considered possible in both groups. The observed slight inter-group variations in the mean organ weights of the Fl female animals are considered not to be attributable to treatment.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Although colour changes of the kidneys are regularly observed in this strain of rats, the increased incidence of yellowish kidneys in group 4 (1800 ppm) is considered treatment-related (males and females).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
A few microscopic changes were observed however they were considered to be incidental findings or background pathology
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Indices did not reveal any differences between dose groups and control.
Dose descriptor:
NOEL
Effect level:
300 ppm
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
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Slight retardation of hair growth and eye opening in high dose group; physical development of low and mid dose group was similar to control
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Pup losses of mid and high dose groups were slightly higher than control (reported to be minimal however significance not indicated)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights reduced in mid and high dose groups (significant on days 1, 4, 14 and 21 post-partum for high dose group and significant on days 4 and 21 for the mid dose group)
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:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Low incidence of malformations and abnormalities were observed however, these types of malformations are known to occur spontaneously in rats and due to their low incidence, they were considered incidental
Histopathological findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
In the F1a generation, results of the employed functional tests underlined or are considered likely to underline the observed retardation of pup development at 1800 ppm (group 4), respectively, but did not reveal any additional treatment-related effect. In the F1b group, functional tests did not show any differences between dose groups and control group.
Developmental immunotoxicity:
not examined
Dose descriptor:
NOEL
Generation:
F1a
Effect level:
300 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
Dose descriptor:
NOEL
Generation:
F1b
Effect level:
300 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Slight retardation of hair growth and eye opening in high dose group; physical development of low and mid dose group was similar to control
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
In the F2a generation, the lower mean numbers of pups found alive on day 1 post-partum in groups 3 (900 ppm) and 4 (1800 ppm) cannot definitively be related to treatment as the differences to the control group were small.
In the F2b generation, the moderately reduced mean number of pups found alive on day 1 post-partum in group 4 (1800 ppm) is considered treatment-related.
During lactation, F2a pup losses from day 1 to 4 post-partum were slightly increased in groups 3 (900 ppm) and 4 (1800 ppm). In the F2b generation, pup losses were slightly increased in group 3 (900 ppm) and markedly increased in group 4 (1800 ppm). These findings are considered attributable to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean pup weight was slightly reduced in low dose group, slightly to moderately reduced in mid dose group, and markedly reduced in high dose group (significant on days 14 and 21 for all groups however, only mid and high dose groups effects were definitively related to 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:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Low incidence of malformations and abnormalities were observed however, these types of malformations are known to occur spontaneously in rats and due to their low incidence, they were considered incidental
Histopathological findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
In the F2b generation, results of the employed functional tests underlined or are considered likely to underline the observed retardation of pup development at 1800 ppm (group 4), respectively, but did not reveal any additional treatment-related effect. In the F2a pups, functional tests did not show any differences between dose groups and control group.
Developmental immunotoxicity:
not examined
Dose descriptor:
NOEL
Generation:
F2a
Effect level:
300 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
Dose descriptor:
NOEL
Generation:
F2b
Effect level:
300 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
Reproductive effects observed:
not specified

Table 1: Overview on indices

  P-generation                           F1-generation                          
  1rst mating            2nd mating            1rst mating            2nd mating           
 Dose (ppm) 0 300 900 1800   0 300 900 1800   0 300 900 1800   0 300 900 1800  
Mating performance (d) 2.4 2.5 2.9 2.5   96.7 96.7 93.3 100   2.4 2.7 2.6 2.6   2.3 2.1 2.8 1.8  
Insemination index (%) 96.7 93.3 100 96.7   96.7 96.7 93.3 100   100 95.8 96 100   92 95.8 100 100  
Fecundity index 96.6 100 96.7 100   89.7 79.3 85.7 90   88 100 87.5 96   73.9 91.3 80 88  
Fertility index 93.3 93.3 96.7 96.7   86.7 76.7 80 90   88 95.8 84 96   68 87.5 80 88  
Gestation index 100 100 100 100   100 100 95.8 96.3   100 100 100 100   100 100 95 100  
  F1a-generation            F1b -generation            F2a -generation            F2b -generation           
 Dose (ppm) 0 300 900 1800   0 300 900 1800   0 300 900 1800   0 300 900 1800  
Live birth index (%) 98.4 98.3 92.5 90.9   96 98.6 95.6 95.4   98.3 93.8 90.2 92.6   97.6 98.2 97.3 92.6  
Weaning index (%) 59.4 59.8 49.1 47.8   89.2 75.7 59.7 47.6   80.4 84.8 75.5 74.8   64.7 68.1 59.6 36.8  
Viability index (d4-1) 96.9 94 89.5 81.2   94.7 96.6 86.2 80.2   93.1 96.9 80 78.2   92.9 93.9 76.3 65.9  
Viability index (d7-4) 96.4 84.8 80.8 80.5   87.5 96.2 80.1 74.5   89.3 91.8 82.5 83.9   83.8 87.5 76.2 58.4  
Viability index (d7-14) 62.3 66.1 64.4 58.3   76.8 78.8 75.6 61.5   85.3 93.8 87.8 89.7   72.2 78.7 76.4 56.3  
Viability index (d14-21) 91.8 93.5 91.6 94.8   94.9 96.6 98 93.7   96.8 98 98.5 97.9   98.8 95.7 94.7 85.2  

Table 2: Group mean body weight (g) of females during first lactation (calculated from animals with live pups on day 21 pp) 

Parameter 0 ppm (group 1)   300 ppm (group 2)   900 ppm (group 3)   1800 ppm (group 4)   ANOVA
Day of lactation N mean SD   N mean SD   N mean SD   N mean SD   F-probability
1 24 305.2 23.1   23 299.1 20.5   24 306.6 19.9   22 285 22.2 (1,2,3) 0.0141
4 24 312.3 28.1   23 306.1 22.1   24 310 18.2   22 291.8 24.7 (1,2,3) 0.02
7 24 315.8 29   23 312 22.7   24 311.7 21.8   22 298.4 24.3   0.0994
14 24 331.7 27.8   23 325.9 20   24 325 18.7   21 312.9 24.1   0.0561
21 24 325 25.4   23 322.6 20.7   24 322.7 21.1   22 320.9 22.9   0.9433
(gr n) = group number which is significantly different from the marked group; p < 0.05, analysis of variance followed by Newman-Keuls test
Table 3: Group mean daily food consumption (g/d) of females during first lactation (calculated from animals with live pups on day 21 pp)       
Parameter 0 ppm (group 1)   300 ppm (group 2)   900 ppm (group 3)   1800 ppm (group 4)   ANOVA
Day of lactation N mean SD   N mean SD   N mean SD   N mean SD   F-probability
1 24 30.5 5.6   23 31.7 4.7   24 29.8 5.3   22 29.4 5.9   0.5132
4 24 35.4 8.2   23 36.6 5   24 33.6 4.8   22 32.8 4.9   0.1337
7 24 45.1 10.4   23 45.7 6.5   24 39 7.6 (1,2) 22 37.7 5.8 (1,2) 0.0006
14 24 56.8 17.6   23 57.4 14.8   24 44.7 12.9 (1,2) 22 43.5 9.2 (1,2) 0.0004
21 24 45.6 11   23 46.3 7.6   24 38.8 7.6 (1,2) 22 37.8 5.8 (1,2) 0.0004
(gr n) = group number which is significantly different from the marked group; p < 0.05, analysis of variance followed by Newman-Keuls test

Table 4 : Litter weights and pup weights of the F1a generation                       
Parameter 0 ppm (group 1)   300 ppm (group 2)   900 ppm (group 3)   1800 ppm (group 4)   ANOVA
  N mean SD   N mean SD   N mean SD   N mean SD   F-probability
                               
Number of pups day 1 pp 28 12.5 2.4   28 12.5 2.2   29 12 3.2   29 11.7 2.6   0.6014*
Litter weight day 1 pp 28 80 14.9   28 77.8 12.4   29 73.5 19.9   29 70.5 16.6   0.1219
Pup weight day 1 pp 28 6.4 0.5   28 6.3 0.6   29 6.1 0.6   29 6 0.6 (gr.1) 0.0761*
                               
Number of pups day 4 pp 28 12.1 2.2   28 11.6 2.7   29 11.1 3.6   29 9.8 3.2 (Gr 1,2,3) 0.0164*
Litter weight day 4 pp 28 98.5 21.5   28 93 27.1   28 84.9 26.1   28 73.7 25.2 (gr 1,2) 0.0023
Pup weight day 4 pp 28 8.2 1.2   28 7.9 1.4   28 7.3 1.3 (gr1) 28 7.2 1.3 (gr1) 0.0108*
                               
Number of pups day 7 pp 28 7.6 1   28 6.8 2.3   29 6.1 2.8 (gr1) 29 5.9 2.6 (gr1) 0.0119*
Litter weight day 7 pp 28 89.3 27.8   27 84.9 33.6   25 77.3 28.6   26 67.8 30.6   0.0568
Pup weight day 7 pp 28 11.7 2.9   27 11.5 3   25 10.7 2.6   26 9.8 2.6   0.0563*
                               
Number of pups day 14 pp 28 4.9 3   28 5 3.1   29 4 2.9   29 3.7 2.8   0.194*
Litter weight day 14 pp 25 148.7 81.3   23 158.2 66.3   24 114.7 72.3   21 102.1 56.4 (gr 2) 0.0243
Pup weight day 14 pp 25 25.5 5.5   23 25.1 5.1   24 22.8 5.2   21 20.3 4.9 (gr 1,2) 0.0008*
                               
Number of pups day 21 pp 28 4.7 3.1   28 4.8 3.1   29 3.6 2.8   29 3.5 2.8   0.1865*
Litter weight day21 pp 24 246.8 130.1   23 254.4 113.5   24 171.3 115 (gr1,2) 22 147.6 82.1 (gr 1,2) 0.0022
Pup weight day 21 pp 24 42.3 8.4   23 41.6 7.9   24 36.4 8.3 (gr1,2) 22 30.6 7.9 (Gr 1,2,3) 0.0001*
                             
pp = post partum                               
(gr n) = group number which is significantly different from the marked group; p < 0.05, analysis of variance followed by Newman-Keuls test     
* Based on taking the ranks of the variables                           

Table 5 : Viability data for F1a generation                             
Parameter 0 ppm (group 1)   300 ppm (group 2)   900 ppm (group 3)   1800 ppm (group 4)   ANOVA
  N mean SD   N mean SD   N mean SD   N mean SD   F-probability
                               
Number of pups day 1 pp 350   349   349   341    
Mean number per female 28 12.5 2.4   28 12.5 2.2   29 12 3.2   29 11.7 2.6   0.7019
                               
Number of pups day 4 pp 338   326   320   284    
mean number per female day 4 pp 28 12.1 2.2   28 11.6 2.7   29 11.1 3.6   29 9.8 3.2 (Gr 1,2) 0.0162
Viability index % (day 4-1) 28 96.9 4.8   28 94 15.8   28 89.5 20.2   28 81.2 23.5 (gr 1,2) 0.0049
                               
Number of pups after adjustment of litter size 220 219 216 213
Mean number per female 28 7 0.8 28 7.8 0.9 29 7.4 1.8 29 7.3 1.7
Number of pups day 7 pp 212   190   176   172    
mean number per female day 7 pp 28 7.6 1   28 6.8 2.3   29 6.1 2.8 (gr1) 29 5.9 2.6 (gr1) 0.0119
Viability index % (day 7-4) 28 96.4 8.9   28 84.6 28.9   28 80.8 31.8   29 80.5 28.8 (gr1) 0.0373
                               
Number of pups day 14 pp 137   139   115   107    
mean number per female day 14 pp 28 4.9 3   28 5 3.1   29 4 2.9   29 3.7 2.8   0.194
Viability index % (day 14-7) 28 62.3 36.4   27 66.1 37   25 64.4 33.3   26 58.3 34.7   0.7383
                               
Number of pups day 21 pp 132   134   105   102    
mean number per female day 21 pp 28 4.7 3.1   28 4.8 3.1   29 3.6 2.8   29 3.5 2.8   0.1865
Viability index % (day 21-14) 25 91.6 23   23 93.5 17.2   24 91.6 15.6   22 94.8 10.3   0.8883
                               
pp = post partum                               
(gr n) = group number which is significantly different from the marked group; p < 0.05, analysis of variance followed by Newman-Keuls test       
* Based on taking the ranks of the variables                             

Table 6: Sex ratio data for F1a generation                         
                     
Parameter 0 ppm (group 1)   300 ppm (group 2)   900 ppm (group 3)   1800 ppm (group 4)
males:females pp day 1 50.3 : 49.7   50.4 : 49.6   50.4 : 49.6   49.9 : 50.1
males:females pp day 21 49.2 : 50.8   49.3 : 50.7   53.3 : 46.7   52:48:00
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
21 mg/kg bw/day
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The test article was administered to rats in feed for 90 consecutive days. In this study, reproductive organs were analyzed by macroscopical and histopathological investigations. Treatment-related adverse effects on reproductive organs were not observed. According to a BAuA research report, effects on reproduction are not expected if repeated dose studies show neither relevantly elevated testis or ovary weights nor histopathological alterations in those organs (BAuA Forschungsbericht Fb 984, 2003). Therefore, the test substance is considered to have no potential to interfere with reproductive performance.

In addition, the data obtained from a structural analogue is also taken into account based on read across approach (see attached read across justification document).

Data from a structurally related compound:

A 2-generation study in rats was performed under GLP following OECD testing guideline 416 with the following deviations: The estrous cycle was not determined prior to mating and sperm parameters were not examined. No adjustment in feed concentration was made during gestation and lactation, so that the dose applied to dams during lactation was strongly increased – for the period of highest feed intake it was doubled. Groups of 30 male and 30 female rats (parent generation) and groups of about 25 male and 25 female rats (F1-generation) of the Sprague-Dawley strain were given 0, 300, 900 and 1800 ppm of the substance mixed to the diet. Treatment was continuous throughout mating, pregnancy, lactation and maturation. The doses were selected based on the results of dose-range finder study.

No mortality and no clinical symptoms occurred with the adult animals of the parental and filial generations. In general, effects on parental animals are consistent with the results of the subchronic toxicity studies with rats. There was no effect of treatment on mating performance and fertility of the animals of the P and F1 generation in any of the three dose groups tested. Histopathology of the reproductive organs showed no test-item related findings.

300 ppm is considered to represent the NOEL (no observed effect level). This dose did not elicit any toxic effects on males and females of the P and F1 generation or on their offspring. It corresponds approximately to a 21 to 26 mg/kg/day dose level if calculated as the overall mean of the weekly mean values of mean daily test article intake (the overall mean of P and F1 males corresponds to approximately 21 mg/kg/day dose level - range of the weekly mean values 14.6 to 47.3 mg/kg/day - and the overall mean of P and F1 females corresponds to approximately 26 mg/kg/day dose level if the gestation and lactation periods are excluded - range of the weekly mean values 20.5 to 47.6 mg/kg/day).

Adverse effects on parental animals were found for food consumption and body weight with females being more sensitive than males. In addition, in the F1 generation, treatment at the high dose level possibly elicited lower mean spleen weight and moderately reduced mean kidney weight and elicited an increased incidence of (yellowish) colour changes in the kidneys in male animals. In the female animals there was an increased incidence of colour changes in the kidneys.

Prior first mating and during first gestation, mean daily food consumption of the female animals of the highest dose group was slightly reduced in comparison with the control. This is reflected in a slightly lower mean body weight gain, the difference being statistically significant during weeks 2 and 7 of treatment. In the other dose groups, mean daily food consumption and body weight gain was comparable with the control during this period.

During first lactation, mean daily food consumption was dosage-relatedly reduced in the mid and high dose group and comparable with the control in the low dose group. Differences between the low and high dose group and the control group were statistically significant from day 7 to 14, 14 to 21, and - consequently - 1 to 21 post-partum.

Prior and during second mating and during second gestation, mean daily food consumption was slightly reduced in the high dose group and comparable with the control in the other two dose groups.

During second lactation, mean daily food consumption was moderately reduced in the high dose group; differences to the control group were statistically significant from day 7 to 14, 14 to 21, and - consequently - 1 to 21 post-partum. In the other dose groups, mean daily food consumption was comparable with the control group during this period.

Since the concentration in the feed was not adjusted for food intake during lactation, exposure to the test item was almost doubled. As a result, lactating dams showed lower body weights at the highest dose group during the first week of lactation and dams of the mid and high dose reduced their food intake during the second and third week of lactation. This correlates with the highest sensitivity of offspring during the first week of lactation and with the absence of treatment-related effects on offspring during the third week of lactation and thereafter.

A reproducible effect during lactation was observed for all four matings for offspring with a NOEL of 300 ppm. For F1a and F1b pups, slightly increased F1a pup loss, possibly related to treatment, and reduced mean F1a pup weight were recorded. For F2a and F2b pups, there was a reduced mean number of F2a pups found alive on day 1 postpartum which cannot definitively be related to treatment, slightly increased F2a and F2b pup loss during lactation, reduced mean weight of F2a and F2b pups and slightly delayed hair growth and eye opening of F2a pups.

Mean pup weight at 1800 ppm was slightly to moderately reduced until day 14 post-partum and markedly reduced on day 21 post-partum, differences to the control group were statistically significant on days 1, 4, 14, and 21 post-partum. In the mid dose group, mean pup weight was slightly reduced until day 14 post-partum and moderately reduced on day 21 post-partum, differences to the control group being statistically significant on days 4 and 21 post-partum. The effects on rat pups during lactation are unlikely to be relevant for human health hazard assessment. For one reason, higher interim maternal toxicity occurred due to doubled dosing during lactation. In addition, in contrast to monkeys and dogs, rats are particularly sensitive to this substance as it affects the pituitary-liver-thyroid axis. The reduced body weight gain of pups is consistent with the reduced body weight of parental animals and the overall toxicity profile in rats.

In summary, no adverse effects to reproductive organs haven been observed on the available 90 day study conducted in male and female rats. In addition, the results of a 2-generation-study with a structurally related compound in rats by the oral route gave no indication of a fertility impairing effect. The NOEL was determined to be 21 - 26 mg/kg bw/day based on dosage and effects during lactation. Based on these results, the test article is not considered to be toxic to reproduction and classification is not warranted.

Effects on developmental toxicity

Description of key information

The results of a teratogenicity study with a structurally related compound in rats (gavage) demonstrated reduced body weight gain of the dams. As a consequence progeny of these dams showed diminished body weight and retardation of the ossification as part of a general retardation of development. 100 mg/kg bw was considered to be a NOEL for both dams and fetuses. The substance is considered to be not teratogenic and not embryotoxic.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Jan. 25, 1982 to Jul. 7, 1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
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 and environmental conditions:
TEST ANIMALS
- Source: From a closed breeding colony (CIBA GEIGY, WST)
- Age at study initiation: 2 months old
- Weight at study initiation: 190 g
- Fasting period before study: Not reported
- Housing: Makrolon cages equipped with a wire mesh top and water bottles, and saw dust (granular form) serving as bedding material. The cages were placed in movable racks kept in a air-conditioned room.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21ºC ± 2ºC
- Humidity (%): 55% ± 10%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 h/12 h
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: Not applicable
Vehicle:
peanut oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The suspension of the test material was freshly prepared daily by means of a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Batches of diet were assayed for composition and contaminant level by the manufacturer. No other details were provided.
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1 Male/3 Females
- Length of cohabitation: Overnight
- Proof of pregnancy: Sperm in vaginal smear referred to as Day 0 of pregnancy
Duration of treatment / exposure:
Day 6 until Day 15 of pregnancy, inclusive.
Frequency of treatment:
Once/day
Duration of test:
Females were killed on Day 21 of pregnancy.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
900 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 rats/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In order to determine the dose levels for the main study, a preliminary experiment was carried out on 10 fertilized rats each for the vehicle control and the dose group (1,000 mg/kg). The test material was suspended in a mixture of distilled water and polyethylene glycol 400 (1:1) and administered orally by intubation from Day 6 until Day 15 of pregnancy, inclusive. Treatment at this 1,000 mg/kg dose level caused a reduction in bodyweight gain and food consumption in the mother animals. Apart from a reduced body weight no adverse effects were recorded for the progeny at sacrifice shortly before term in comparison with the vehicle control. On the basis of the foregoing results the doses for the main study were selected at 0, 100, 300 and 900 mg/kg of body weight.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the period of treatment, general body condition, weight gain and symptoms were checked daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During the period of treatment, general body condition, weight gain and symptoms were checked daily.

BODY WEIGHT: Yes
- Time schedule for examinations: The average body weight gain of the females was determined on the basis of the actual body weight as well as that on the first day of treatment and at the time of necropsy, corrected by subtracting the weight of the gravid uterus.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No applicable

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: Ovaries and uterus (mucosa and contents, including amniotic fluid and placentae as well as abortions and resorption sites)
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
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes
- Skeletal examinations: Yes: two-thirds of the litter
- Head examinations: Yes
Statistics:
Chi-square test, Yates' correction, Student's t-test (one-tailed)
Indices:
None reported.
Historical control data:
Spontaneous data characteristic of the breed of rats used in the present study refer to a series of untreated controls ("historical" or cumulative control) observed over a period of about 5 years.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Females No. 1, 4 (vehicle control) and 50 (100 mg/kg) died spontaneously on days 12, 11 and 8 p.c. (i.e. days 7, 6 and 3 of treatment).
respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The dams of the intermediate and high-dose groups reacted to the treatment by a reduction in body-weight gain.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The dams of the intermediate and high-dose groups reacted to the treatment by a reduction food consumption
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:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
The numbers of embryonic and/or fetal deaths (resorptions) were not increased at either dose
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw (total dose)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The progeny of the intermediate and high-dose groups displayed diminished body-weight shortly before term.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male to female ratios of the foetuses were also comparable for all groups.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal maturation showed some delay in these groups: phalangeal nuclei, calcanei and sternebrae were affected in this respect; in the 900 mg/kg group the number of still incompletely ossified thoracic vertebral centres was found to be enhanced, in addition. The occurrence of sternebral anomalies in all groups was not assumed to be of an experimental significance, the overall incidence of sternebral anomalies being 0.26% in the historical control.
Visceral malformations:
no effects observed
Description (incidence and severity):
One occasional malformation found in the high-dose group (generalized oedema) was considered to be of a spontaneous origin and not related to the treatment. Generalized oedema occurred at a rate of 0.02% in the historical control population of the breed of rats used for the present experiment.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
other: some delay in skeletal maturation
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The substance was devoid of embryotoxic activity and did not reveal teratogenic potency in the rat under the present experimental conditions. Some delay of physiological growth was noted for the foetuses of a 300 mg/kg and, in particular, the foetuses of the 900 mg/kg group but considered to be non-specific and related to maternal toxicity; the low-dose (100 mg/kg) was unaffected in this respect.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
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

Data from the structurally related compound (see attached read across justification document)

The available study was performed under GLP and following the procedure outlined in OECD testing guideline 414 (2001). It is adequate in design and reporting detail to allow for hazard assessment. In order to determine the dose levels for the main study, a preliminary experiment was carried out on 10 fertilized rats each for the vehicle control and the dose group (1000 mg/kg). The test material was suspended in a mixture of distilled water and polyethylene glycol 400 (1:1) and administered orally by intubation from day 6 until day 15 of pregnancy, inclusive. Treatment at this 1000 mg/kg dose level caused a reduction in bodyweight gain and food consumption in the mother animals. Apart from a reduced body weight no adverse effects were recorded for the progeny at sacrifice shortly before term in comparison with the vehicle control. On the basis of the foregoing results the doses for the main study were selected at 0, 100, 300 and 900 mg/kg of body weight. The test material was suspended in peanut oil and administered once daily by the oral route from day 6 until day 15 of pregnancy, inclusive.

The dams and the progeny of the 100 mg/kg dose group remained unaffected by the treatment. The dams of the intermediate and high-dose groups reacted to the treatment by a reduction in body-weight gain and food consumption. In a close relation to these findings, the progeny displayed diminished body-weight of the fetuses examined shortly before term. Skeletal maturation showed some delay in these groups: phalangeal nuclei, calcanei and sternebrae were affected in this respect; in the 900 mg/kg group the number of still incompletely ossified thoracic vertebral centers was found to be enhanced, in addition.

In conclusion, the substance was devoid of an embryotoxic activity and did not reveal teratogenic potency in the rat under the present experimental conditions. Some delay of physiological growth was noted for the fetuses at 300 mg/kg, in particular, the fetuses of a 900 mg/kg group but considered to be non-specific and related to maternal toxicity; the low-dose (100 mg/kg) was unaffected in this respect.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for reproduction toxicity is not warranted under Regulation (EC) No.1272/2008.