Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because a pre-natal developmental toxicity study is available
Cross-reference
Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June - August 1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted prior to GLP similar to OECD guideline 414 on the registered substance itself. The method is to be considered scientifically reasonable with minor deficiencies in documentation.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not applicable
GLP compliance:
no
Remarks:
Test conducted prior to GLP implementation
Limit test:
no
Species:
rat
Strain:
Long-Evans
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BAYER AG, breeding FB 30 (in-house)
- Age at study initiation: 2.5 - 3.5 months (females), 3 - 6 months (males)
- Weight at study initiation: 192 - 254 g (females), 350 - 500 g (males)
- Fasting period before study: no
- Housing: except for mating, dams were housed individually in Makrolon type II cages under conventional conditions; for mating two females and one male were housed over night in Makrolon type III cages
- Diet (e.g. ad libitum): pelletted Altromin-R feed ad libitum
- Water (e.g. ad libitum): tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23°C
- Humidity (%): ca. 60%
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
400 (Lutrol)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 0, 0.1%, 0.3%, 0.9%
- Amount of vehicle (if gavage): 5 ml/kg bw
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male / 2 females
- Length of cohabitation: 1 night
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
10 days, from gestation day 6-15
Frequency of treatment:
daily
Duration of test:
from mating to gestation day 20
Remarks:
Doses / Concentrations:
0, 5, 15, 45 mg/kg bw /day
Basis:
other: nominal amount
No. of animals per sex per dose:
25 females / dose
Control animals:
yes, concurrent vehicle
Details on study design:
The structurally related substance Imidazolidinthion-2 (Ethylenthiourea, ETU) was similarly applied at a dose of 45 mg/kg bw/day as positive control.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes

BODY WEIGHT: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Uterus
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes, a certain number was examined regarding thoracic and abdominal organs
- Skeletal examinations: Yes, a certain number was examined
- Head examinations: Yes: all per litter for external malformations and sceletal anomalies
Statistics:
Testing for statistically significant differences of treatment groups compared to control was performed using the following methods:
- Distribution-free WILCOXON rank-sum test (Wilcoxon-Mann-Whitney-U-Test) for body weight gain of females, number of implantations, resorptions, foetuses per litter, average weight of foetuses per litter and average placenta weight per litter
- Chi-Square-Test (YATES corrected) for number of needy forms as well as foetuses with bone alterations resp. malformations per litter
- Chi-Square-Test (dependent on expected frequencies), YATES corrected, or as so-called FISHER’s exact test, for the rate of inseminated and pregnant animals.
Calculations were performed in BAYER computing centre Leverkusen with an analysis program developed for embryotoxicity testing. As long as not noted otherwise, a difference is statistically significant if the probability value is below 5% (p < 0.05).
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: slightly diminished body weight gain of dams

Details on maternal toxic effects:
See "Any other information on results incl. tables"
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
45 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: no dose- or substance-related effects

Details on embryotoxic / teratogenic effects:
See "Any other information on results incl. tables"
Dose descriptor:
NOAEL
Effect level:
>= 45 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
>= 45 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Effects on the dams

 

Morbidity

 

Rats of the solvent control were all healthy and did not show any changes in appearance and behaviour during the whole course of the test.

In the treatment groups, the following individual observations were made:

 

Table 1: Individual observations in treatment groups

Number of females with

Group

Diarrhoea

Coryza

Bloody vagina

Accelerated Breathing

Slight ataxia

White urine with sediments

Abscess in uterus

Control

-

-

-

-

-

-

-

5 mg/kg

5

-

-

-

-

-

-

15 mg/kg

1

2

-

-

-

-

-

45 mg/kg

1

-

1

1

1

1

-

45 mg/kg ETU

5

1

-

-

-

-

-

 

These individual observation do not allow any attribution to the applied substance or dose and are likely to be caused by infections

 

 

Mortality

 

None of the animals dies during the course of the study.

 

 

Body weight gain during pregnancy

 

The average changes in body weight are summarized in Table 2:

 

Table 2: Body weight gain of the dams

Group

Body weight gain / g

During treatment

Total pregnancy

Control

62.8

149.7

5 mg/kg

62.4

152.4

15 mg/kg

57.8

143.7

45 mg/kg

44.0

133.3

45 mg/kg ETU

52.0

144.6

 

Body weight gain of pregnant rats is slightly reduced after gavage of either 45 mg/kg bw/day N-Methyl-2-thion-thiazolidin or ETU

 

 

Results of insemination and impregnation

 

Of the inseminated rats the following number were impregnated resp. pregnant at the day of the caesarean section:

 

Table 3: Results of insemination and impregnation

Substance

Dose / mg/kg

Inseminated rats

Impregnated rats

Pregnant rats

Total

%

Total

%

Control

n/a

25

23

92.0

23

100.0

Methyl-2-thion-thiazolidin

5

25

23

92.0

23

100.0

Methyl-2-thion-thiazolidin

15

25

22

88.0

22

100.0

Methyl-2-thion-thiazolidin

45

25

24

96.0

24

100.0

ETU

45

25

25

100.0

25

100.0

 

The rates of impregnated and pregnant treated animals does not differ significantly from control. Slight differences are in the range or normal biological variations.

 

 

Effects on intrauterine development

 

Embryolethality

 

None of the tested substances resp. doses had an embryolethal effect. Slight differences in resorption rate are in the range or normal biological variations.

 

 

Embryotoxicity and growth retardation

 

Methyl-2-thion-thiazolidin did not show any growth-retarding or embryotoxic effects up to highest dose tested. So-called needy forms, i.e. foetuses weighing < 3g on day 20 of pregnancy, were observed in the following numbers:

 

Table 4: Number of observed needy forms

Group

Dose

Number

Remarks

Control

n/a

10

In 3 litters

Methyl-2-thion-thiazolidin

5 mg/kg bw/day

7

In 4 litters

Methyl-2-thion-thiazolidin

15 mg/kg bw/day

0

 

Methyl-2-thion-thiazolidin

45 mg/kg bw/day

14

In 5 litters

ETU

45 mg/kg bw/day

59

In 10 litters

 

It was noticed that needy forms mainly appeared in litters in which also malformations occurred, in both control and treated animals.

 

 

Teratogenicity

 

In both control and treated animals malformations occurred as set out in Table 5.

 

Table 5: Observed malformations

Group

Dam No.

Number of foetuses with malformations

Type of malformation

Control

2382

4

3x Telencephalonhypoplasia

3x Telencephalonhypoplasia

 + brain ventricle dilatation

 + shortening of phalanges

2401

4

4x Telencephalonhypoplasia

2452

4

4x Telencephalonhypoplasia

Methyl-2-thion-thiazolidin, 5 mg/kg

2430

3

3x Tail kink

2495

4

3x Telencephalonhypoplasia

1x Telencephalonhypoplasia

 + Microphthalmia

Methyl-2-thion-thiazolidin, 15 mg/kg

2494

1

1x Hydrops, Sternum dysplasia, dysplasia of all long bones, general abnormality of the extremities

Methyl-2-thion-thiazolidin, 45 mg/kg

2385

2

2x Telencephalonhypoplasia

2431

3

3x Telencephalonhypoplasia

2456

4

3x Telencephalonhypoplasia

1x Telencephalonhypoplasia

 + Microphthalmia

2479

3

3x Telencephalonhypoplasia

 

The in the groups treated with Methyl-2-thion-thiazolidin observed Telencephalonhypoplasia are certainly not connected to the treatment as they were observed to the same extent in the control groups. This partial hypoplasia of the CNS is most probably a spontaneous mutation in this breeding line of FB30 rats. This was shown over a longer period also in other studies, but vanished after switching to another breeding line.

5 – 45 mg/kg bw/day Methyl-2-thion-thiazolidin are, solved in Polyethylene glycol 400, and applied by gavage during gestation day 6 to 15 not teratogen in Long-Evans rats.

 

After gavage of 45 mg/kg bw/day ETU the percentage of foetuses with minor aberrations of ossification was doubled. All foetuses in this group were malformed. Malformations affect very uniformly the neurocranium, extremities as well as spine and tail. Here, there were observed hydrocephaly, encephalomeningoceles, meningoceles, asymmetries and changes in shape as well as partial agenesis of the spine, tail kink and, regarding extremities, besides misalignments and so-called cross-legged posture, also oligo-, ektro- and syndactyly. Further, approximately half of the foetuses showed malformations on the ribs (missing, floating, fused, shortened ribs and cusp formation). With minor occurrence there were hydromyelia in the spine and spina bifida, cleft palate, abnormities on the jaws, hydronephrosis and hydroureter as well as sporadic aplasia, ectopic testicles as well as cryptorchism and sporadic open eyes observed. The distribution of the malformations is shown in Table 6.

 

Table 6: Distribution of malformations

Group

Dose / mg/kg

Number of examined foetuses

Number of malformed foetuses

Number of pregnant rats

Number of dams with malformed foetuses

n

%

Control

0

265

12

4.5

23

3

Methyl-2-thion-thiazolidin

5

270

7

2.3

23

2

Methyl-2-thion-thiazolidin

15

251

1

0.4

22

1

Methyl-2-thion-thiazolidin

45

277

12

4.3

24

4

Imidazolidinthion

45

299

299

100

25

25

 

Conclusions:
The study was conducted prior to GLP similar to OECD guideline 414 on the registered substance itself. The method is to be considered scientifically reasonable with minor deficiencies in documentation. Hence, the results can be considered as sufficiently reliable to assess the developmental toxicity of N-Methyl-2-thion-thiazolidin. There were no treatment-related effects in developmental parameters. The developmental NOAEL is ≥ 45 mg/kg bw/day, the highest dose tested, where already slight maternal toxicity was observed. Hence, N-Methyl-2-thion-thiazolidin does not need to be regarded as developmental toxicant.
Executive summary:

In a developmental toxicity study (equivalent to OECD 414) N-Methyl-2-thion-thiazolidin was administered to 25 inseminated female Long-Evans rats/dose by gavage at dose levels of 0, 5, 15, or 45 mg/kg bw/day from days 6 through15 of gestation.

 

None of the animals died during the course of the study. After gavage of 45 mg/kg bw/day, a slightly diminished body weight gain of dams, and a tendency of growth retardation in foetuses was observed. 15 mg/kg bw/day were tolerated without any adverse effects.

 

The maternal LOAEL is 45 mg/kg bw/day, based on slightly diminished body weight gain of dams, and tendency of growth retardation in foetuses. The maternal NOAEL is 15 mg/kg bw/day.

 

There were no treatment-related effects in developmental parameters.

 

The developmental LOAEL could not be determined. The developmental NOAEL is ≥ 45 mg/kg bw/day, the highest dose tested.

 

The developmental toxicity study in the rat is classified as acceptable, and satisfies sufficiently the guideline requirements for a developmental toxicity study (OECD 414) in rats.

Data source

Materials and methods

Results and discussion

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion