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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study similar to OECD 414 guidelines before GLP statement (1979). Pregnant animals were treated from day 7 to day 16 gestation instead of day 5 to day 15 corresponding to organogenesis in rodents. Historical control data were not reported.
Cross-reference
Reason / purpose:
reference to other study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1979
Reference Type:
publication
Title:
Unnamed
Year:
1987

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
The animals were treated from the day 7 to the day of 16 instead of the first day of gestation up to the end of gestation; i.e. two days of organogenesis were not included (organogenesis in rodents is between day 5 to 15 of gestation).
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Remarks:
study conducted before GLP
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Hexamethylene Diamine (HMD)
- Physical state: liquid
- Substance type: diamine
- Lot/batch No.: 2487
- Purity: 85.8% w/w aqueous solution
- Composition of test material, percentage of components: no data
- Stability under test conditions: no data
- Storage condition of test material: no data

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Wilmington, Mass.
- Age at study initiation: ten-week old
- Weight at study initiation: 176-225 g for females
- Fasting period before study: no data
- Diet: ad libitum (Wayne Lab Chow Mash)
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): light/dark cycle of 14 hour light / 10 hour dark

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test solutions were prepared by diluting an 85.8% w/w aqueous solution of HMD to concentration of 112, 184 and 300 mg/10 L with distilled water.

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food:no data

VEHICLE
- Concentration in vehicle: 112, 184 or 300 mg / 10 mL
- Amount of vehicle (if gavage): 10 mL/kg b.w./day
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1M/2F or 1M/1F
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy
- Any other deviations from standard protocol: no data
Duration of treatment / exposure:
From day 7 to day 16 of gestation
Frequency of treatment:
daily
Duration of test:
From the first day to day 22 of gestation (sacrifice) including HMD treatment from dat 7 to day 16 of gestation.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 112, 184 and 300 mg/kg b.w./day
Basis:
other: Range finding preliminary study (see Preache, 1979 a)
No. of animals per sex per dose:
22 pregnant rats/dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: based on the findings in a range-finding study (Data indicated that the maximum dosage in the teratogenicity study should be above 225 mg/kg bw/day in order to reach a clear maternally toxic level. Dose levels of 400 and 900 mg/kg bw were lethal. Concerning the low dosage for the teratogenicity study, based the lack of effect on the incidence of embryonic or fetal deaths, or on fetal measurements and external morphology at 112.5 mg/kg bw/day would suggest that a dosage at approximately this level should be satisfactory.)
- Rationale for animal assignment (if not random): not applicable
- Other: no data

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: determined on days 7-16 and 22 of gestation.


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 data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 22
- Organs examined: laparectomized for exposure of the uterus and ovaries.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number if live and dead fetus: Yes
Fetal examinations:
- External examinations: Yes: all live fetus per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
Statistics:
The incidence of specific maternal observations was analysed by the Fisher Exact Probability Test or the Chi-square Test as appropriate (Siegel, 1956All data that were in the form of a percentage of occurence within a litter were transformed by the inverse sine transformation prior to application of analysis of variance (Steel and torrie, 1960). Where the analysis of variance yielded a significant F ratio, treated group means, based on transformed data where appropriate, were individually compared to that of the control group by Dunnett's t test (Steel and Torrie, 1960). A level of significance of p < 0.05 was used throughout.
Indices:
No data
Historical control data:
Not reported

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: at the highest dose

Details on maternal toxic effects:
There was one maternal death in each of the 0, 184 and 300 mg/kg bw/day groups and one animal in the latter group that was sacrified prior to term because she appeared moribund.
Maternal body weight gain was reduced as compared to controls for animals administered 300 mg/kg bw/day of HMD after the first treatment and remained suppressed throughout gestation.
Other evidence of maternal toxicity at this dosage was a transient decrease in food consumption and a decrease in the number of dams that were free of toxic signs during daily clinical observations.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
ca. 184 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Neither pre- nor post-implantation survival was adversely affected by treatment with HMD.
Fetal body weight was reduced by treating the dams at 300 mg/kg/day of HMD but lower dosages had no effect on the body weights of the fetuses nor was fetal crown-rump lenght or the distribution of surviving male and female fetuses affected by HMD at any of the dosage levels.
No external observations indicated a significant difference between the control and treated groups. In the visceral examinations, the average percentage of fetuses having virtually no liver spottiness was found to be less in the 300 mg HMD/kg bw/day group than in control fetuses.
Overall the incidence of minor and major malformations was low and not significant at 184 mg/kg bw/day. Although a variety of anatomical variations and ossification delays were observed in animals dosed at 184 mg/kg bw/day, these were seldom restricted to the fetuses of treated animals as the control animals showed the same kind of malformations.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 7.8.2/1: Summary by dose level: Maternal Mortality, Body Weight, and Body Weight Change

Dosage

(mg/kg bw/day)

Females Placed on Test

No. Of Females Pregnant

% Of Females Pregnant

No. Of Pregnant females that Dieda

% Of Pregnant Females that Died

Body Weight Day 7 (g)b

Body Weight Change (g)b

Day 8

Day 9

Day 10

0

22

21

95.5

1

4.8

273 ± 13

2+5c

5+4c

8+5c

112

22

22

100.0

0

0

271 ± 12

0+4

4+5

8+4

184

22

22

100.0

1

4.5

274 ± 10

-2+5

0+7

4+7

300

22

22

100.0

2

9.1

266 ± 11

-8+8d

-14+11d

-14+14d

aThe only nonpregnant females survived to necropsy

bMean ± standard deviation

c F ratio significant, p < 0.05

d Significantly different from 0 mg/kg/day dosage group, p < 0.05

Table 7.8.2/2: Body Weight Change

Dosage

(mg/kg bw/day)

Body Weight Change (g)

Day 11

Day 12

Day 13

Day 14

Day 15

Day 16

Day 22

Adjusted

Day 22

0

12 ± 7c

18 ± 6c

22 ± 6c

26 ± 6c

31 ± 8c

36 ± 10c

110 ± 29

20 ± 8c

112

13 ± 5

16 ± 5

22 ± 5

26 ±5

31 ± 6

37 ± 6

109 ± 16

19 ± 10

184

7 ± 8

11 ± 8

17 ± 9

21 ± 8

26 ± 7

32 ±7

108 ± 9

17 ± 8

300

-12 ± 15d

-8 ± 15d

0 ± 16d

4 ± 15d

9 ± 18d

22 ± 14d

98 ± 20

6 ± 12d

c F ratio significant, p < 0.05

d Significantly different from 0 mg/kg/day dosage group, p < 0.05

Table 7.8.2/3: Summary by dosage level: Maternal Food Consumption

Dosage

(mg/kg bw/day)

Food Consumption (G, Mean + SD) for Intervals Ending

Day 4

Day 7

Day 10

Day 13

Day 16

Day 19

Day 22

0

55 ± 9

59 ± 7

56 ± 7a

62 ± 6a

63 ± 10

70 ± 23

66 ± 7

112

54 ± 7

58 ± 4

54 ± 9

60 ± 5

65 ± 6

75 ± 20

65 ± 9

184

53 ± 9

59 ± 7

50 ± 7

55 ± 9b

62 ± 10

70 ± 7

67 ± 7

300

53 ± 9

58 ± 7

44 ± 12b

44 ± 12b

58 ± 8

75 ± 10

64 ± 5

a F ratio significant, p < 0.05

b Significantly different from 0 mg/kg/day dosage group, p < 0.05

Table 7.8.2/4: Summary by dosage level: Embryonic and Fetal Loss and Fetal Measurements

Dosage (mg/kg bw/day)

Number of

% Preimplantation Lossab

No. of Live Fetuses

% Male Fetusesab

% Dead or Resorbed Fetusesab

Fetal Body Weight (g)a

Fetal Crown-Rump Distance (mm)a

Litters

Corpora Lutea

Implantationsa

Male

Female

Male

Female

0

20

16 ± 3

14 ± 4

11.4 ± 23.0c

14 ± 4

50.1 ± 17.4

3.8 ± 4.7

5.62 ± 0.38c

5.34 ± 0.35c

42 ± 2

41 ± 1

112

22

15 ± 2

15 ± 2

3.4 ± 6.3

14 ± 2

51.6 ± 11.6

7.6 ± 8.7

5.39 ± 0.33

5.09 ± 0.32

42 ±2

41 ± 2

184

21

15 ± 2

15 ± 2

2.4 ± 5.0

14 ± 1

50.8 ± 16.6

5.1 ± 6.6

5.35 ± 0.37

5.15 ± 0.33

42 ± 1

41 ± 2

300

20

15 ± 1

15 ± 2

0.3 ± 2.6d

14 ± 2

49.6 ± 11.4

2.4 ± 4.2

5.18 ± 0.55d

4.96 ± 0.54d

42 ± 2

41 ± 2

aMean/litter ± standard deviation

bMeans and standard deviations shown are based on untransformed data; for analysis of variance percentages were transformed by the inverse sine transformation

cF ratio significant, p < 0.05

d Significantly different from 0 mg/kg/day dosage group, p < 0.05

Applicant's summary and conclusion

Conclusions:
Under the test conditions, maternal toxicity was observed at 300 mg/kg bw/day of HMD with secondary maternal toxicity effects observed in the fetuses. However, no adverse effect for maternal toxicity was observed at 184 mg HMD/kg bw/day and no reliable developmental effect was observed at 300 mg/kg bw/d.

In conclusion, no classification is required for HMD as demonstrated in the developmental study.
Executive summary:

A preliminary study determined that the hexamethylene diamine (HMD) maximum dosage in the teratogenicity study should be above 300 mg/kg b.w/day in order to reach a clear maternally toxic level. The dose range was selected to include maternal toxicity and no effect dosage

The lack of effect in the incidence of embryonic or fetal deaths, or on fetal measurements and external morphology observed at 112.5 mg/kg b.w./day in the preliminary study suggested that a dosage at approximately this level should be satisfactory in the teratogenicity.

A study similar to OECD Guideline 414 (Prenatal Developmental Toxicity Study), pregnant female Sprague-Dawley rats (22/group) were treated by gavage with different concentrations of diluted 85.8% w/w aqueous solution of HMD at dosage level of 0, 112, 184 and 300 mg/kg b.w./day on the days 7-16 of gestation.

Prenecropsy observations were carried out such as body weight changes, food consumption, and cage side observation. Dams that survived to day 22 of gestation were killed on that day and necropsy was performed. Finally, fetals examinations were realized. The maternal parameters assessed during the study included body weight, feed consumption, clinical signs, corpora lutea, implantations and resorptions. The fetal parameters assessed during the study included litter size, placental weight, gross malformations, fetal crown-rump length, fetal body weight, sex, visceral and skeletal examinations.

One maternal death occurred in each of the 0, 184 and 300 mg/kg b.w./day groups and one animal in the latter group that was sacrified prior to term because she appeared moribund. Maternal body weight gain was reduced as compared to concurrent controls for animals treated at 300 mg HMD/kg b.w./day throughout the gestation. Other evidence of maternal toxicity at this dosage was a transient decrease in food consumption and clinical observations such as hunching, kemps fur, red stained fur, wheezing and respiratory rattle.

Litter size was comparable in all groups. Fetal body weight was reduced by treating the dams with 300 mg/kg b.w./day of HMD but lower dosages had no effect on the body weights of the fetuses. Sex ratio, crown-rump length and percent dead or resorbed fetuses were unaffected by the treatment. Pre-implantation loss was significantly lower in the high-dose group, but high in the control group.

Treatment with HMD at a dosage of 300 mg/kg b.w./day on days 7-16 of gestation induced maternal toxicity as evidenced by reduced body weight gains, transiently decreased food consumption, clinical observations, and by the death of approximately 10% of the animals treated at this dosage level. Dosage below 300 mg/kg b.w./day had no statistically significant effects on the dams; however, the initial dosing at the mid dose level caused a transient mean body-weight loss of 2 grams on day eight of gestation. At the maternal toxicity dose level of 300 mg HMD/kg b.w./day, the fetuses were slightly retarded in development as evidenced by body weight, limited retardation of skeletal development, and possibly by liver spottiness.

A dosage of 112 mg/kg b.w./day clearly had no effect on the fetuses including poorly ossified cervical vertebral centra frequency.

The frequency of occurrence of fetuses with poorly ossified cervical vertebral centra was significantly greater than for concurrent controls in both the 184 and 300 mg HMD/kg b.w./day dosage groups and for the latter group, there were significantly more fetuses in which the sacral and caudal vertebra had unfused components. According to the authors, both these observations are consistent with a slight retardation in skeletal development and considered as a ¿fingerprint¿ of a generalized delay in ossification in near term rats (sacrifice on the gestation day 22). Although the delayed of ossification observed in the fetuses from the dams treated at the highest dose were secondary to the maternal toxicity, the retardation in skeletal development observed at the middle dose level occurred in the absence of maternal toxicity.

The generalized delay is characterized by reduced ossification of bones that normally exhibit rapid ossification during the last few days of gestation such as the cervical, sacral and caudal vertebral centra. In rodents, while bones such as ribs and long bones of the limbs ossify early, other bones such as thoracic and lumbar vertebral centra are among the regions that ossify rapidly during late gestation.

Historical control data provide another means of characterizing the normal pattern of skeletogenesis, and are extremely important for interpreting delayed ossification. Moreover, the laboratory-specific data are needed to interpret delayed ossification data because the criteria for scoring criteria for certain bones are so detailed that the historical control incidence of delayed ossification often approached 100%. This situation suggests that the lab has not discriminated between the normal range of variation in skeletal ossification and variation beyond the normal range making the usefulness of the observations questionable. Hence, in the absence of the historical control data (along with concurrent control) in this study, it should be impossible to determine the designation and the occurrence of the poorly ossified cervical vertebral centra in the context of the background ¿noise¿ of the population on test or at risk.

Another common but questionable practice is to conduct independent statistical analyses on different degrees of ossification for a single bone (e.g., for cervical vertebral centra ¿ unossified, poor ossified, unarticulated or bilobed). Although these distinctions can be identified readily by experienced technicians from a developmental perspective, they are of minimal significance. Problem can arise when these isolated findings are interpreted solely on the basis of statistical significance rather than considering the overall context of closely related variations. In effect, the incidence of incompletely ossified vertebral centra may be increased statistically, yet the unarticulated and bilobed vertebral centra were decreased as demonstrated in this study. Hence, the interpretation based solely on the statistically identified increase has led to the inappropriate conclusion that ossification of the cervical vertebral centra was delayed, when in fact, consideration of the cervical vertebral centra data as a whole would indicate the lack of an adverse effect.

Hence, the slight ossification retardation observed in the study at both highest dose level is considered to be of low level of concern as nonlethal and not detrimental to postnatal survival variations which are generally reversible or transitory.

This teratogenicity study is considered as acceptable. It does satisfy the guideline requirement for a teratogenicity study for an OECD 414 guideline in the rat. Hence, this study can be considered as valide for classification.

Under the test conditions, no adverse effect for maternal toxicity was observed at HMD dose of 184 mg/kg bw/day and no reliable developmental effect was observed as 300 mg/kg bw/d. In conclusion, no classification for HMD is required as demonstrated by the results observed in the developmental study.