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Key value for chemical safety assessment

Effects on fertility

Description of key information

The NOEL for the test material was determined to be 3 mg/kg bw/day according to a two generation rat study performed in line with EPA Guideline OPP 83-4.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 July 1987 to 21 April 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
Deviations:
yes
Remarks:
(actual relative humidity was 20-94%; three incorrect pups were selected for the F1 generation; in addition, the following protocol deviation was also reported; on three occasions test diets were prepared in advance and stored frozen)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: (P) approxaimtely 6 wks; (F1) nominally 4 wks
- Weight at study initiation: (P) Males: 139.7-217.4 g; Females: 107.7-149.9 g;
- Housing: stainless steel wireless cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 40-70 %
- Air changes: minimum 15 per hour
- Photoperiod: 12 hours light/12 hours dark
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly (excpet on three occassions when the diet was prepared in advance and frozen)
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 15 days
- Proof of pregnancy: sperm in vaginal smear
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken from the diet formulations prepared in weeks 1, 13, 26 and 36 and analysed by shaking samples with hexane and water (low dose groups) or hexane only (high dose groups). Hexane solutions are further diluted and analysed using GC.
Duration of treatment / exposure:
Two generations
Frequency of treatment:
Daily
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
Dose / conc.:
60 ppm
Dose / conc.:
350 ppm
Dose / conc.:
2 000 ppm
No. of animals per sex per dose:
Thirty (P); twenty-five (F1 generation)
Control animals:
yes, plain diet
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly except for mated females which were weighed on days 0, 6, 12, 15 and 20 of gestation and days 1, 4, 7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes (weekly)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (sex distribution as equal as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
stillbirths, live births, postnatal mortality, litter weights, and clinical condition of pups

GROSS EXAMINATION OF DEAD PUPS:
yes
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals (at time of weaning of F1 generation).
- Maternal animals: All surviving animals (at time of weaning of F1 generation).

GROSS NECROPSY
- Gross necropsy was performed.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were removed: ovaries, uterus, cervix, vagina, lesions, pituitary, testes, epididymides, seminal vesicles, prostate and coagulating gland.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at time of weaning.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy was performed.
- Number of implantation sites were counted and recorded for F1 females.
Statistics:
Using nested analysis of variance for normally distributed errors or by non-parametric techniques for non-normally distributed errors.

The standard deviation obtained from analysis of variance was used for 't' tests between control and treatment groups. Where necessary, data were transformed before analysis.

Non-parametric testing was performed using the Kruskal-Wallis test for a treatment-related response.

Significant differences between control and treatment groups were determined using the Wilcoxon rank sum test.
Reproductive indices:
Median pre-coital time = time by which half the females had mated

Fertility index = (no. of pregnant females / no. of paired females) x 100

Fecundity index = (no. of pregnant females / no. of mated females) x 100

Gestation index = (no. of females with live pups / no. of pregnant females) x 100
Offspring viability indices:
Post-implantation survival index (F1 only) = (no. of pups born / no. of implantation sites) x 100

Live birth index = (no. of pups alive on day 1 / no. of pups born) x 100

Viability index 1 = (no. of pups alive on day 4 before culling / no. of pups alive on day 1) x 100

Viability index 2 = (no. of pups alive on day 7 / no. of pups alive on day 4 after culling) x 100

Viability index 3 = (no. of pups alive on day 14 / no. of pups alive on day 7) x 100

Viability index 4 = (no. of pups alive on day 21 / no. of pups alive on day 14 x 100)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical observations of P generation adults were restricted to those commonly seen in this strain of rat in these laboratories e.g. occasional instances of rough hair coat, slight hair loss, fur staining and chronnodacryorrhoea. Incidence of fur staining and slight hair loss was slightly increased at 2000 ppm in P generation females during late gestation.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Treatment did not adversely affect mortality. One P generation male was found dead during week 14; necropsy revealed a liver mass. This death was not attributable to treatment. There were no other adult deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains were lower in males and females at 2000 ppm; body weight gain in females during gestation was also lower at 2000 ppm.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was lower than controls in males and females at 2000 ppm. Initial lowering of food consumption in P generation animals was so marked as to suggest adverse palatability of the test material in the diet.
Food efficiency:
no effects observed
Description (incidence and severity):
Food conversion efficiency was similar in all groups, with the exception of week 1 for the highest dose level.
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
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy findings of parents were not adversely affected by treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no significant histopathological effects on either the pituitary or reproductive tract attributable to test material administration.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance, fertility, fecundity, pregnancy rate and duration of gestation were not adversely affected by treatment.
Key result
Dose descriptor:
NOEL
Effect level:
60 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: equivalent to 3 mg/kg bw/day Based on lower bodyweight gain in offspring in both adults and pups, no effects on fertility were noted
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations of F1 generation adults were restricted to those commonly seen in this strain of rat in these laboratories e.g. occasional instances of rough hair coat, slight hair loss, fur staining and chronnodacryorrhoea.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Offspring survival was not adversely affected by treatment. One F1 generation male was killed during week 12 following accidental injury to the snout. This death was not attributable to treatment. There were no other adult deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains were lower in males and females at 2000 ppm; body weight gain in females during gestation was also lower at 2000 ppm. Body weight gain was lower than controls in F1 males at 350 ppm.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was lower than controls in males and females at 2000 ppm.
Food efficiency:
no effects observed
Description (incidence and severity):
Food conversion efficiency was similar in all groups.
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:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy findings of weaned offspring were not adversely affected by treatment.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no significant histopathological findings for either the pituitary or reproductive tract attributable to test material administration. Hyaline droplet nephropathy was recorded in the kidneys of three high dose male. This finding was considered related to test material administration.
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
60 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: equivalent to 3 mg/kg bw/day Based on lower bodyweight gain in offspring in both adults and pups, no effects on fertility were noted
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No observed adverse effects
Critical effects observed:
not specified
Reproductive effects observed:
not specified

Table 1: Summary of adult performance

   Dose group (ppm)
   0   60  350  2000
   P  F1  P  F1  P  F1  P  F1
 Males                        
 In group  30  25  30  25  30  25  30  25
 Dead: post pairing  0  0  0  0  0  0  1  1
 Failing to induce pregnancy  1  -  2  -  2  -  1  -
 Females                        
 In group  30  25  30  25  30  25  30  25
 Pregnant/%  29/96.7  25/100  28/93.3  20/80  28/93.3  24/96  29/96.7  25/100
 With total resorption  1  -  0  -  0  -  0  -
 Total litter loss  4  4  3  4  3  4  5  2
 With live pups at day 21 post-partum  24  21  25  16  25  20  24  23

Table 2: Group mean body weight gain (g)

     Dose group (ppm)
     0  60  350  2000
 Time interval  Sex  P  F1  P  F1  P  F1  P  F1
 0 -5  M  199.9  260.6  192.0  256.7  192.7  242.6  148.1  201.9
   F  90.5  120.5  91.9  122.1  88.0  111.5  67.1  98.7
 6 -10  M  88.9  104.6  98.8  113.6  83.5  93.8  68.8  71.0
   F  32.1  40.3  31.0  37.7  34.5  39.4  23.8  33.3
 11 -13  M  33.2  44.7  23.3  43.8  29.5  37.6  -6.1  41.7
   F  100  112  109  115  109  108  88  85
 14 -16  M  29.8  31.8  29.3  41.8  30.7  48.3  53.7  42.6
   F  29  46  40  36  53  45  65  60

*14-17 weeks for males of the F1 generation

Table 3: Group mating data

   Dose group (ppm)
  0  60  350  2000
 Mating indices  P  F1  P  F1  P  F1  P  F1
 Number of paired females  30  25  30  25  30  25  30  25
 Number of mated males  30  25  30  22  29  24  29  25
 Number of pregnant females  29  25  28  20  28  24  29  25
 Fertility index (%)  96.7  100  93.3  80  93.3  96  96.7  100
 Fecundity index (%)  96.7  100  93.3  90.9  96.6  100  100  100

Table 4: Group mean pup weights (g)

   Dose group (ppm)
   0  60  350  2000
 Day  P  F1  P  F1  P  F1  P  F1
 1 (combined)  6.1  5.7  5.7  5.9  5.5  5.9  5.6  5.8
 4 (combined)  8.6  7.9  7.9  8.4  7.2  7.7  6.9  7.8
 7 (combined)  13.8  13.2  13.1  13.7  12.1  12.8  10.4  12.0
 14 (combined)  29.8  30.0  29.1  29.8  27.4  27.4  21.9  24.4
 21 (combined)  49.8  50.9  49.5  49.7  46.7  45.98  38.2  39.9
 21 (males)  51.2  51.8  50.3  51.0  49.1  47.2  39.5  40.6
 21 (females)  49.6  49.6  48.7  48.5  44.7  45.9  37.9  38.9
 % weight changes  716  793  4768  742  749  678  582  588

Table 5: Group incidence histopathology findings

    P generation F1 generation
    Dose group* and sex Dose group* and sex
    0 ppm M 2000 ppm M 0 ppm F 2000 ppm F 0 ppm M 2000 ppm M  0 ppm F 2000 ppm F
Animals examined     30  30  30  30  25**  24**  25**  25**
Animals unremarkable     10  10  19  23  8  9  15  17
Skin Acanthosis  0  1  3  3  1 91)  1 (2)  9 (9)  7 (7)
  Dermatitis  2  0  3  0  0 (1)  1 (2)  2 (9)  0 (7)
  Adnexal atrophy  -  -  -  -  0 (1)  0 (2)  0 (9)  1 (7)
Liver Necrosis (lobar)  0  1  0  0  1 (1)  -  -  -
Kidneys Focal nephropathy  0  10  0  0  -  -  -  -
  Hydroneophrosis  2  0  0  0  1 (1)  1 (3)  -  -
  Nephroblastoma  0  0  0  1  -  -  -  -
  Hyaline droplets  -  -  -  -  0 (1)  3 (3)  -  -
  Basophilic tubules  -  -  -  -  0 (1)  3 (3)  -  -
  Casts  -  -  -  -  0 (1)  3 (3)  -  -
  Cyst  -  -  -  -  0 (1)  1 (3)  -  -
Pituitary Altered cell focus  0  2  0  0  -  -  -  -
  Cyst  0  3  0  0  1  0  0  0
  No sample  0  0  1  0  -  -  -  -
Oral cavity Necropsy finding/ no equivalent finding  2  1  2  2  -  1 (1)  -  -
  Stomatitis  -  -  -  -  -  1 (1)  -  -
Nasal cavity Rhinitis  -  -  -  -  -  1 (1)  -  -
Abdominal cavity Necropsy finding/ no equivalent finding  0  1  0  0  -  -  -  -
Lungs Inflammatory cell foci  -  -  -  -  1 (1)  -  -  -
  Congestion/heamorrhage  2  1  0  1  1 (1)  -  -  -
  Foamy histiocytes  2  1  0  0  -  -  -  -
  Lymphoid hyperlasia  1  1  0  0  -  -  -  -
  Pneunomitis  2  1  0  0  -  -  -  -
Skull Exostoses  1  1  0  0  -  -  -  -
Foot Arthritis  1  0  0  0  -  -  -  -
  Dermatitis  0  1  0  0  -  -  -  -
Tail Dermatitis/folliculitis  0  3  0  0  1 (1)  -  -  -
Mandiubular lymph node Hyperplasia  -  -  -  -        
Ear Chondritis  -  -  -  -        
Testis Atrophy  1  1  -  -  2  2  -  -
  Sperm granuloma  -  -  -  -  0  1  -  -
Epididymis Inflammatory cell foci  0  1  -  -  1  0  -  -
  Oligospermia  0  1  -  -  0  2  -  -
Seminal vesicle Not remarkable  30  30  -  -  24  25  -  -
Coagualting gland One sample  4  3  -  -  0  1  -  -
Protstate Inflammatory cell foci  -  -  -  -  6  6  -  -
  Prostatitis  10  10  -  -  3  4  -  -
Ovary Not remarkable  -  -  30  30  -  -  25  25
  One sample  -  -  -  -  -  -  1  0
Uterus Arteritis  -  -  1  0  -  -  0  0
  Oestrus stage  -  -  1  1  -  -  0  0
Cervix Not remarkable  -  -  -  -  -  -  0  0
Vagina Not remakrable  -  -  30  30  -  -  0  0
Cause of death Liver lesion    1            
  Upper respiratory infection/ stomatitis            1    

If less animals were examined, the real number examined is presented between brackets.

Conclusions:
Under the conditions of the test, administration of the test material to rats over two generations at dietary concentrations up to 2000 ppm did not adversely affect adult reproduction or offspring survival. Body weight gains were lower in adults and offspring at 2000 ppm and to a lesser extent at 350 ppm. Food consumption was also reduced in adults at 2000 ppm. No adverse effects of treatment were seen at the low level of 60 ppm which is considered to be the NOEL (equivalent to 3 mg/kg bw/day).
Executive summary:

In a GLP compliant study conducted in line with standardised guideline EPA OPP 83-4, the effect of the test material on reproductive toxicity was determined in the rat.

Rats were administered the test material by oral (diet) at 0, 60, 350 and 2000 ppm.

Adult reproduction or offspring survival was not adversely affected. Body weight gains were lower in adults and offspring at 2000 ppm and to a lesser extent at 350 ppm. Food consumption was also reduced in adults at 2000 ppm. No adverse effects of treatment were seen at the low level of 60 ppm which is considered to be the NOEL (equivalent to 3 mg/kg bw/day).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
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

A two-generation reproduction toxicity study in rats with dose levels up to 2000 ppm did not adversely affect adult reproduction or offspring survival. Body weight gains were lower in adults and offspring at 2000 ppm and to a lesser extent at 350 ppm. Food consumption was also reduced in adults at 2000 ppm. No adverse effects of treatment were seen at the low level of 60 ppm; this dose therefore constitutes a NOEL (corresponding to 3 mg/kg bw/day).

Effects on developmental toxicity

Description of key information

The NOAEL for both maternal and embryo/foetotoxicity for the test material was determined to be 45 mg/kg bw/day according to a study performed in line with OCED Guideline 414 and EPA Guideline OPP 83-3.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 March 1988 to 15 August 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
18 mated females instead of 20 were used in the study
Qualifier:
according to
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
18 mated females instead of 20 were used in the study
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 3.00-4.48 kg at mating
- Housing: individually in stainless steel cages
- Diet: ad libitum
- Water: mains water ad libitum
- Acclimation period: at least 19 days before mating

ENVIRONMENTAL CONDITIONS
- Temperature: 16-22 °C
- Humidity: 40-70 %
- Air changes: minimum of 15 per hour
- Photoperiod: 14 hours light/10 hours dark
Route of administration:
oral: gavage
Vehicle:
other: 1 % gum tragacanth
Details on exposure:
Suspensions of the test material in vehicle were prepared weekly and dispensed as daily aliquots
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test formulation were taken from the first and second weekly preparations for analysis.
Details on mating procedure:
Each female was mated with one proven stud New Zealand White buck.
Duration of treatment / exposure:
To mated females from days 7 to 19 of gestation.
Frequency of treatment:
Once daily
Duration of test:
Animals were killed on day 29 of gestation.
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
45 mg/kg bw/day (actual dose received)
Dose / conc.:
135 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
18
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during gestation

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 7, 13, 19, 23 and 28 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined: days 0-7, 7-13, 13-19, 19-23, and 23-29 of gestation.
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 and intrauterine position of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: pregnancy status
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]
Statistics:
Levene's test was used to test for equality of variances between groups for body weight gain (except days 7-13), food intake, uterus weight and mean litter weight. Where there was a significant difference between groups using one-way analysis of variance, pairwise t-tests between treated groups and the control were conducted. Where Levene's test was significant (p<0.05), non-parametric tests were performed.

Major and minor external/visceral defects and major skeletal defects were analysed by comparing the proportion of litters in the treated groups with one defect to the proportion in the control group using the Fisher exact test.

Kruskal-Wallis non-parametric analysis of variance was performed on all other parameters. Where there was a significant overall difference between groups (p<0.05), the Wilcoxon Rank Sum Test was performed for each treated group against the controls.
Indices:
Percentage pre-implantation loss = ((no. of corpora lutea - no. of implantation sites) / no. of corpora lutea) x 100

Percentage of post-implantation loss = ((no. of implantation sites - no. of live young) / no. of implantation sites) x 100

Females as % of males = (no. of females / no. of males) x 100

Fertility index = (no. of females pregnant / no. of females mated) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
The clinical condition of surviving animals was generally comparable in all groups.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Four of the treatment animals died or were killed, two in the low dose and one in both the median and high dose groups. The distribution between the treated groups did not indicate an effect of treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean body weight gain of the high dose animals was significantly lower than the controls. The majority of high dose animals lost weight from day 7 to day 13 of gestation. In three animals the loss persisted until treatment ceased. There was no adverse effect of treatment on weight gain at the low or intermediate dose level.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake of high dose animals was significantly lower than controls over the treatment period. No significant differences were observed at the low and intermediate dose levels.
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:
no effects observed
Description (incidence and severity):
Necropsy revealed no treatment-related lesions.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Necropsy revealed no treatment-related lesions.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
not examined
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
The number of corpora lutea and implantations per dose were comparable in all groups. Pre-implantation loss was within the expected range in all groups. Post-implantation loss was higher than controls in the high dose group. The increase was mainly attributable to two atypical litters (number 66 and 72) and was not statistically significant. The number of foetuses per litter was correspondingly reduced. The incidence of intrauterine deaths in the low and intermediate dose groups was lower than controls and litter size was marginally increased.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
not examined
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The number of pregnant animals surviving to day 29 of gestation was between 14 and 16 in every group.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy incidence was 100 % in the low dose and 94.4 % in all other groups.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
45 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Foetal weight was lower than controls in the high dose group. The reduction was not statistically significant and was attributable to three atypical litters (number 66, 71, and 72). There was no adverse effect on foetal weight in the low and intermediate dose groups.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
The incidence of litters containing foetuses showing major defects was significantly higher than controls in the high dose group. Again the increase was mainly attributed to litters 66, 71, and 72 suggesting that these were mediated by maternal toxicity. There was no adverse effect of treatment on the incidence of major defects in the low or intermediate dose groups. The incidence of minor defects or variations did not indicate an effect of treatment at any of the dose levels investigated.
Skeletal malformations:
not specified
Visceral malformations:
not specified
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
45 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
45 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Mean weight (kg), body weight gain (kg) and food intake (g)

     Dose group (mg/kg bw/day) 
 Day  Parameter  0  15  45  135
 0  Body weight  3.63  3.61  3.76  3.68
 7  Body weight  3.78  3.77  3.91  3.84
   Body weight gain  0.15  0.16  0.15  0.16
   Food intake  201  204  212  212
 13  Body weight  3.83  3.84  3.97  3.70***
   Body weight gain  0.05  0.07  0.06  -0.14
   Food intake  175  184  184  115***
 19  Body weight  3.94  3.97  4.08  3.80
   Body weight gain  0.11  0.13  0.11  0.10
   Food intake  168  171  170  124*
 23  Body weight  4.03  4.0  4.16  3.85
   Body weight gain  0.09  0.04  0.08  0.05
   Food intake  148  141  158  132
 29  Body weight  4.16  4.03**  4.23  4.00
   Body weight gain  0.13  0.02  0.07  0.15
   Adjusted body weight  3.58  3.48  3.68  3.51
 Food intake  127  92  118  122
 Total mean  Body weight gain  0.53  0.42  0.47  0.32
   Food intake  166  161  171  144

*P<0.05; ***P<0.001

Table 2: Group incidence of foetuses with defects

     Dose group (mg/kg bw/day)
 Observation  0  15  45  135
 Number of foetuses examined  142  154  146  115
 External and visceral effects  Major  0  1  1  11**
   Minor  5  14  2  12
   Variants  46  47  70  39
 Skeletal  Major  1  0  0  24
   Minor  33  45  36  16
   Variants  128  141  130  106
 Total number of foetuses showing major defects  1  1  1  26

** P<0.01

Conclusions:
Under the conditions of the test, administration of the test material at 135 mg/kg bw/day elicited maternal toxicity, characterised by maternal weight loss at the onset of dosing and reduced food intake over the treatment period. In three animals, the reaction was so severe as to elicit corresponding foetal effects. The litters of these animals showed reduced foetal weight and an increased incidence of intrauterine deaths and major abnormalities. In the remaining litters, the incidence of foetal effects did not greatly differ from normal values. There was no adverse effect of treatment, maternal or foetal, at 15 or 45 mg/kg/day in either this or the previous study (Barker, 1989b). The NOAEL for both maternal and embryo/foetotoxicity in this study is 45 mg/kg bw/day.
Executive summary:

In a GLP compliant study conducted in line with standardised guidelines OECD 414 and EPA OPP 83-3, the effect of the test material on developmental toxicity was determined in the rabbit.

Rabbits were administered the test material by oral (gavage) at 0, 15, 45 and 135 mg/kg bw/day. Administration of the test material at 35 mg/kg bw/day elicited maternal toxicity, characterised by maternal weight loss at the onset of dosing and reduced food intake over the treatment period. In three animals, the reaction was so severe as to elicit corresponding foetal effects. The litters of these animals showed reduced foetal weight and an increased incidence of intrauterine deaths and major abnormalities. In the remaining litters, the incidence of foetal effects did not greatly differ from normal values. There was no adverse effect of treatment, maternal or foetal, at 15 or 45 mg/kg/day in either this or the previous study (Barker, 1989b).

The NOAEL for both maternal and embryo/foetotoxicity in this study is 45 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 September 1983 to 26 January 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 13 weeks
- Weight at study initiation: 182 to 237 g
- Housing: suspended steel cages
- Diet: stock diet ad libitum
- Water: tap water ad libitum
- Acclimation period: seven days

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 2 °C
- Humidity: >40 %
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Route of administration:
oral: gavage
Vehicle:
other: 1 % tragacanth gum
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Length of cohabitation: overnight
- After 1 day of unsuccessful pairing replacement of first male by another male with proven fertility.
- Breeding continued until the desired number of females was obtained.
Duration of treatment / exposure:
From day 6 up to and including day 15 of pregnancy
Frequency of treatment:
Daily
Duration of test:
Up to day 21 of gestation
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
180 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Twenty five pregnant females
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: general condition and behaviour

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 6, 16 and 21 of pregnancy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined: day 0 to day 6, day 6 to day 16 and day 16 to day 21 of pregnancy
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: foetal length, placenta weight of live foetuses, empty uterus weight
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter] (all groups except 20 mg/kg bw/day)
- Head examinations: No
Statistics:
Student's t-test was used to determine the differences in degree of ossification between test and control groups to transformed ossification values (DgOt) expressed in degrees and calculated by:

DgOt = √DgOº

Differences in body weight, food consumption, organ weights, litter data, foetus weights and lengths and placenta weights were analysed by applying analysis of (co)-variance followed by Dunnett's multiple comparison test whereas skeletal and visceral anomaly were evaluated by Chi-square test.
Indices:
Percentage pre-implantation loss (PRIL) = ((no. of corpora lutea - no. of implantation sites) / no. of corpora lutea) x 100

Percentage of post-implantation loss (POIL) = ((no. of implantation sites - no. of live young) / no. of implantation sites) x 100

Degree of ossification of foetus skeletons (DgO) = no. of bones without ossification (or with incomplete ossification) / no. of bones examined
Clinical signs:
no effects observed
Description (incidence and severity):
No abnormalities in condition or behaviour of the animals were observed.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No deaths of the animals were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Weight gain of animals in the 60 and 180 mg/kg dose group was reduced during the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake of animals in the 60 and 180 mg/kg dose group was reduced during the treatment period.
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):
no effects observed
Description (incidence and severity):
No abnormalities in behaviour of the animals were observed.
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
Other effects:
not examined
Number of abortions:
not examined
Pre- and post-implantation loss:
not examined
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
not examined
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The gestation index was 100 in all groups.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Maternal performance was comparable in all groups and the fertility indices ranged from 84.0 in the high dose, 88.0 in the 60 mg/kg does and 92.0 in both the control and low dose group.
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other: maternal toxicity
Fetal body weight changes:
not specified
Reduction in number of live offspring:
not specified
Changes in sex ratio:
not specified
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
not specified
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal examination of the foetuses did not indicate any embryo/foetotoxic or teratogenic effects that could be related to treatment. The slightly increased incidence of supernumerary thoracic ribs in the 180 mg/kg dose group was not considered to be of toxicological significance.
Visceral malformations:
no effects observed
Description (incidence and severity):
Visceral examination of the foetuses did not indicate any embryo/foetotoxic or teratogenic effects that could be related to treatment.
Other effects:
no effects observed
Description (incidence and severity):
Autopsy findings, organ weights and litter data did not reveal any embryo- or foetotoxic effect of the test material.
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Mean maternal body weight, body weight gain and food intake (g)

     Dose group (mg/kg bw/day)
 Day  Parameter  0  20  60  180
 0  Body weight  202.1  201.7  201.3  204.5
 6  Body weight  226.1  225.1  224.5  229.6
   Body weight gain  24.0  23.4  23.3  25.0
   Food intake  17.3  17.3  17.8  18.3
 16  Body weight  267.0  262.1  253.5**  255.6*
   Body weight gain  40.9  37.0  29.0**  26.0**
   Food intake  19.5  18.2  16.5**  15.4**
 21  Body weight  326.9  323.5  313.1  322.3
   Body weight gain  59.9  60.6  59.6  66.2
   Food intake  21.2  21.6  22.2  23.5**
 Total  Body weight gain  124.8  121.5  111.9  117.9

* P<0.05; **P<0.01

Table 2: Statisitically significant alterations

     Dose group (mg/kg bw/day)
Observation     0  20  60  180
Number of litters examined  23  22  22  21
 umber of foetuses examined  279  272  248  266
Visble alterations Small focal subcutaneous haemorrhage or petachia  0  2  3 (2)  3 (3)
  Dysmature appearance  0  0  0  1
Number of litters examined  23  n.d  22  21
Number of foetuses examined  134  n.d.  114  129
Skeletal alterations Malformations  0  0  0  0
  Minor anomalies  18    10  12
  Variants  25    24  37*
Number of litters examined  23  n.d.  22  21
Number of foetuses examined  134  n.d.  114  129
Ossification asent (in degrees) Forelimb phalanges  65.49    53.55***  72.74*
  Hindlimb phalanges  56.59    53.95  60.11
  Cervical vertebrae bodies  37.99    29.33*  42.28
  Sternebrae  0.59    0  7.03**
Ossification incomplete (in degrees) Forelimb phalanges  12.88    12.31  9.66
  Hindlimb phalanges  17.12    13.72  21.75**
  Cervical vertebrae bodies  26.53    18.07**  22.82
  Sternebrae  10.94    8.75  18.49**
Number of litters examined  23  22  22  21
Number of foetuses examined  145  140  131  135
Visceral Malformations  1  0  0  3 (2)
  Minor anomalies  14 (10)  27* (13)  23 (10)  26* (17)
  Variants  14 (10)  24 (14)  27* (17)  25* (14)

Number of affected litters between brackets

n.d. = not determined

* P<0.05; **P<0.01; ***P<0.001

Conclusions:
Under the conditions of the test, weight gain and food intake in maternal animals was reduced in 60 and 180 mg/kg bw/day dose groups but no effects on fertility indices were seen. Autopsy findings, organ weights, litter data and visceral and skeletal examination of the foetuses did not reveal any embryotoxic, foetotoxic or teratogenic effects that could be related to the test material. The NOAEL for maternal toxicity was determined to be 20 mg/kg bw/day; the NOAEL for embryo/foetotoxicity was determined to be 180 mg/kg bw/day.
Executive summary:

In a GLP compliant study conducted in line with sound scientific principles (and stated as being similar to standardised guideline OECD 414), the effect of the test material on developmental toxicity was determined in the rat.

Rats were administered the test material by oral (intra-oesophagic intubation) at 0, 20, 60 and 180 mg/kg bw/day.

Weight gain and food intake in maternal animals was reduced in the 60 and 180 mg/kg bw/day dose groups but no effects on fertility indices were seen. Autopsy findings, organ weights, litter data and visceral and skeletal examination of the foetuses did not reveal and embryotoxic, foetotoxic or teratogenic effects that could be related to the test material.

The NOAEL for maternal toxicity was determined to be 20 mg/kg bw/day; the NOAEL for embryo/foetotoxicity was determined to be 180 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
45 mg/kg bw/day
Species:
rabbit
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A combined embryotoxicity/teratogenicity study in rats showed that the weight gain and food intake of animals in the 60 and 180 mg/kg dose groups was reduced during the treatment period. The only other effect found was the slightly increased incidence of supernumerary thoracic ribs in the 180 mg/kg group, however this was considered to be of no toxicological significance. The NOAEL for maternal toxicity is 20 mg/kg bw/day and the NOAEL for embryo/fetotoxicity is 180 mg/kg bw/day.

The available teratology studies in rabbits revealed that the administration of the test material at 135 mg/kg bw/day elicits maternal toxicity, characterised by maternal weight loss at the onset of dosing and reduced food intake over the treatment period. The NOAEL for both maternal and embryo/fetotoxicity is 45 mg/kg bw/day.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance is considered to be unclassified for toxicity to reproduction.