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Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1988
Reference Type:
study report
Title:
Unnamed
Year:
1992
Reference Type:
study report
Title:
Unnamed
Year:
1993

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Physical state: solid
- Storage condition of test material: in the dark at ambient room temperature

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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)

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
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

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).