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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Jun 1982 - 22 Jul 1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: OECD 416
Deviations:
yes
Remarks:
Only 3 applications per week; no or only partial examination of oestrous cycle and spermatogenicity performed, no analytical purity reported
Principles of method if other than guideline:
The study was conducted similar to OECD TG 416 (2-generation reproductive toxicity study). Therefore, no continous exposure with the test material was performed during gestation. No fetal examinations are available since pups were used for further mating to produce F2 generation.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Fischer 344
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, Michigan, USA
- Age at study initiation: (P) 6 - 7 weeks; (F1) 4 - 7 weeks
- Weight at study initiation: (P) Males: 119.5 - 142.8 g; Females: 104.4 - 137.4 g; (F1) Males: 57.2 - 162.1 g; Females: 52.8 - 112.7 g
- Housing: animals were housed individually in wire-mesh bottom, polycarbonate cages
- Use of restrainers for preventing ingestion: no data
- Diet: Purina Formulab Chow (#5008), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1
- Humidity (%): 40 - 65
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 Jun 1982 To: 22 Jul 1983

Administration / exposure

Route of administration:
dermal
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: The test material was applied on the back of each animal.
- Time intervals for shavings or clippings: The back hair was shaved each week prior to initial weekly application and when necessary throughout the week to ensure adequate dermal exposure.

TEST MATERIAL
- Amount(s) applied: 1 mL/kg bw
- Concentration (if solution): 1, 10 and 25% (w/v)
- Constant volume or concentration used: yes

VEHICLE
- Amount(s) applied: 1 mL/kg bw

USE OF RESTRAINERS FOR PREVENTING INGESTION: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Since dosing solutions were analysed by a combination of high performance liquid chromatographic method and gas chromatographic method, and were stable for at least 7 weeks, separate batches of dosing solutions were prepared approximately every four weeks. Rats were treated with a new batch of the appropriate dosing solution within 7 weeks. Each dosing solution was analysed prior to its use for verification of the test material´s concentration. The maximum observed deviation from the nominal concentration was 10%.
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 (how): individually in a polycarbonate cage with Absorb-dri (nestwood chips) as nesting material
Duration of treatment / exposure:
(P) Males: 102 days before mating.
(P) Females: 170 days from the initiation of the study until the F1 generation was weaned
(F1) Males: 123 days at weaning, during growth into adulthood, mating and production of an F2 generation
(F1) Females: 221 days at weaning, during growth into adulthood, mating and production of an F2 generation, 30 days after the last litter was weaned
Frequency of treatment:
3/week (except during mating)
Duration of test:
Day 0 of Gestation until weaning
No. of animals per sex per dose:
30 P males, 30 P females
20 F1 males, 40 F1 females
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations were made once daily during treatment and mating periods for morbidity, mortality, general physical appearance, and clinical signs of toxicity. During gestation and lactation periods rats were observed in the morning and afternoon.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Male and female body weights were taken weekly up to the time of mating. Females were weighed on days 0, 7, 14, and 20 of gestation and on days 1, 4, 7, 21 and 28 of lactation. Pups were weighed as a litter on days 1, 4, 7, 21 and 28 of lactation, and were also weighed individually on day 28 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE: No

WATER CONSUMPTION AND COMPOUND INTAKE: No
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: No
Fetal examinations:
- External examinations: No
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
Organ weight, organ weight/body weight ratio, LDH-X enzyme activity and litter size, sex ratio, survival index and gestational length were analysed by analysis of variance. Treatment groups were compared to the control group by one-tailed Dunnett´s t-test at p=0.05. Body weights were analysed by analysis of covariance. Body weights in all dose groups were adjusted for body weight on the first day of dosing due to differences in initial weights among the treatment groups. Gestational weights were adjusted to the common body weight on gestational day 0, and lactational weights were adjusted to that on lactational day 1. Pup weights and number of live and dead pups were adjusted for the differences in litter size among all dose groups. The analysis of covariance was used to adjust the treatment means for the differences in the covariates among the treatment groups. Subsequent treatment comparisons against the control were then made on the adjusted treatment means. The adjusted treatment means represent the means of the treatment groups if they were to have the same mean value in the covariate. Fertility indices were analysed by one-tailed Fisher´s exact test. Mating indices were analysed by Chi-square with continuity correction. Sperm counts and survival indices were analysed by non-parametric method, Kruskal-Wallis test. Treatment groups were compared to the control by one tailed Dunnett´s t-test at p=0.05 on the ranked data.
Indices:
Mating Index (%) = (No. of females mated / No. of females housed with males) x 100
Fertility Index (%) = (No. of females pregnant / No. of females mated) x 100
Survival Index (%) = (pups alive at day 1 / total born pups) x 100
Lactation Index (%) = (pups alive at day 28 / pups alive day 4 after culling) x 100
Historical control data:
no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: non-adverse

Details on maternal toxic effects:
In the F0 adults, body weights of the 1% and 10% dose groups were not affected by the treatment. However, significantly lower (p<0.05) body weights of the 25% dose group were observed during certain intervals when compared to the control. There were statistically significantly lower body weights in the 25% dose group males between days 35-105 during the dosing period; in females, body weights were only significantly lower at day 49, but were slightly lower than the control during the rest of the dosing period. In the F1 adults, body weights of the 1% and 10% dose groups were not affected by the treatment when compared to the control group. However, body weights of the 25% dose group males were lower but only statistically significant (p<0.5) at days 21, 28 and 35 during the dosing period; body weights of the 25% dose group females were lower from days 14-119 during the dosing period when compared to the controls. There were no compound-related effects on maternal body weights of all dose groups during the gestational and lactational periods in the F0 and F1 generations when compared to the controls. On lactational day 14, maternal body weights of the 1%, 10% and 25% dose groups were statistically significantly higher than the control. These increases were of no toxicological significance. In the F1 adult females, significantly higher absolute and relative weights of liver, heart and kidney and the relative lung weight of the 25% dose group, higher absolute and relative heart weights and relative liver weights of the 10% dose group were noted.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
corresponding to 10% (w/v)
Basis for effect level:
other: No toxic effects.
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
corresponding to 25% (w/v)
Basis for effect level:
other: Changes in body weight and organ weights without associated pathological findings.

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: non-adverse

Details on embryotoxic / teratogenic effects:
Two pups, one male from a litter of the F1 10% dose group and one female from another litter of the F2 25% dose group were found dead, and they appeared to have been dehydrated. No pathologic finding was noted in these pups. One female pup (F2 1% dose group) in a litter was born with a short right forelimb and the left hindlimb and was reported missing on day 4 postnatally. Presumably it was canabalised by its mother. Another female pup in a litter (F2 25% dose group) was born dead with a short forelimb and small eyes. The left ocular zone contained a small piece of ocular tissue, the optic cup without a lens; the right eye was smaller than usual. The low incidence of these findings in rats is apparently due to spontaneous occurrence of developmental anomalies. There was no compound related effect on the pup weights of any dose group in either the F1 or F2 generations when compared to the controls. There was no compound-related effect on the development of the pups. All observed effects were considered to be non-adverse.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
corresponding to 25% (w/v)
Sex:
male/female
Basis for effect level:
other: No toxic effects.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean litter size, mean number of live pups and sex ratios of F1 and F2 pups during lactation

Dose concentration in % (w/v)

0

1

10

25

F1 pups

Day 0 - Litter size

8.2

9.6

7.9

6.4

Day 0 Dead pups

0.0

0.1

0.1

0.1

Day 0 Live pups

8.2

9.5

7.8

6.3

Day 1 Live pups

8.2

9.4

7.7

6.3

Day 4 Live pups

8.2

9.4

7.7

6.3

Day 7 (a) Live pups

7.6

8.6

7.0

5.9

Day 14 Live pups

7.6

8.6

7.0

5.9

Day 21 Live pups

7.6

8.6

6.9

5.9

Day 28 Live pups

7.6

8.6

6.9

5.9

Sex ratio of pups % males/%females

46/54

40/60

47/53

45/55

F2 pups

Day 0 - Litter size

6.9

8.3

8.0

8.4

Day 0 Dead pups

0.1

0.0

0.0

0.1

Day 0 Live pups

6.8

8.3

8.0

8.3

Day 1 Live pups

6.7

8.3

7.9

8.2

Day 4 Live pups

6.7

8.2

7.9

8.1

Day 7 (a) Live pups

6.7

7.9 (b)

7.6 (b)

7.8

Day 14 Live pups

6.7

8.1 (b)

7.6

7.6

Day 21 Live pups

6.6

8.0

7.6

7.6

Day 28 Live pups

6.6

8.0

7.4

7.6

Sex ratio of pups % males/%females

46/54

44/56

52/48

50/50

(a): after culling

(b): one litter was excluded from the calculation because the number of live pups were not counted

Table 2: Mean pup body weights (adjusted for litter size) for F1 and F2 pups (a)

Dose concentration in % (w/v)

0

1

10

25

Body weights (g) F1 pups

Day 1

5.40

5.43

5.41

5.62

Day 4

8.10

7.77

7.93

8.56

Day 7

11.75

11.64

11.30

12.35

Day 14

21.42

21.20

22.30

22.48

Day 21

33.72

33.67

32.36

35.33

Day 28

59.06

59.13

58.53

60.62

Body weights (g) F2 pups

Day 1

5.56

5.65

5.66

5.74

Day 4

8.18

8.50

8.68

8.70

Day 7

12.07

12.21

12.53

12.70

Day 14

20.47

21.24

21.52

21.67

Day 21

30.77

31.80

32.78*

32.32

Day 28

55.89

57.76

59.34

57.81

(a): mean pups were calculated from litter weights divided by number of pups per litter

*: significantly different from the control group by using a one-tailed Dunnett´s t-test at p≤0.05

Applicant's summary and conclusion