Reaction mass of hydroxyethyl-DETA, DETA and dihydroxyethyl-DETA

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not applicable

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline and GLP study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

No. 421 draft

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diethylenetriamine (DETA)

IUPAC Name:

Diethylenetriamine (DETA)

Constituent 2

Reference substance name:

2,2'-iminodi(ethylamine)

EC Number:

203-865-4

EC Name:

2,2'-iminodi(ethylamine)

Cas Number:

111-40-0

Molecular formula:

C4H13N3

IUPAC Name:

N-(2-aminoethyl)ethane-1,2-diamine

Test material form:

other: Clear yellow liquid

Details on test material:

- Name of test material (as cited in study report): Diethylenetriamine (DETA)
- Physical state: Clear yellow liquid
- Analytical purity: 98.9%
- Lot/batch No.: F286E6QL79
- Expiration date of the lot/batch: 31 July 2016
- Storage condition of test material: Room temperature, protected from light, with nitrogen

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Ninety-one virgin female, and 95 male Wistar W.U. albino rats of about 6 weeks old, bred under SPF-conditions, were obtained from Charles River Wiga, Sulzfeld, Germany. Upon arrival, the animals were taken in their unopened shipping boxes to a quarantine room, and checked for overt signs of ill health and anomalies. Also a serological control was conducted. Once the results of the serology tests were known and satisfactory, the animals were first moved to study room. In this room they were acclimatized to the animal room conditions for a period of 14 days (preliminary study) or 30 days (reproduction study) .

HOUSING
Upon arrival in the study room, males and females were housed in groups of four per sex per group in suspended stainless steel cages (45x32x18c m) with wire mesh floor and front.
Preliminary study:
The animals assigned to the preliminary study were housed in groups of four per sex per group in similar stainless steel cages (45x32x18 cm).

Reproduction study:
During the premating period of the reproduction study, the animals were housed individually in similar but smaller cages (18x32x18 cm). During mating the animals were housed in pairs of either sex belonging to the same dose group in these cages (18x32x18 cm). Upon evidence of mating, males and females were again housed individually. Around day 18 of gestation the females were moved to macrolon cages (45x32x18 cm) with a wire-mesh top and were provided with sawdust for the remainder of the gestation period and for the lactation period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 and 23.5
- Humidity (%): 55 and 70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: multipore filtered water

Details on exposure:

Diethylene triamine was administered daily by gavage to male and female Wistar rats at levels of 30, 100 and 300 mg/kg b.w. during a 2-week premating period, and during mating and gestation up to day 4 post partum or at least during a 4-week period, to screen its effect on male and female reproductive performance and on the development of the offspring .

Details on mating procedure:

Premating exposure period (male and female): 2 weeks

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis for the stability of the test substance in the vehicle showed that DETA was stable in the vehicle for at least 10 days when stored at approx. 4ºC. The content of the samples taken from the solutions actually given to the animals showed that generally the concentrations of DETA were close to the nominal values.

The reference solutions and the samples were analysed using a Perkin Elmer 8500 gas chromatograph equipped with a Perkin Elmer AS-100 B autosampler.

Duration of treatment / exposure:

29-54 days

Frequency of treatment:

Once per day

Details on study schedule:

The test substance solutions were administered to male and female rats during the entire study. After a 2 week premating period, the males and females were paired 1:l within the same dose groups. Vaginal smears were taken the next morning to determine whether mating had occurred. If no sperm cells were detected, the female in question was placed again with the male. Upon the presence of sperm in the smear or the finding of a vaginal plug, the females were housed individually, and this moment was considered as day 0 of gestation. This procedure was continued until the females were sperm-positive, or 2 weeks had elapsed.

At the end of the study, the non-pregnant females were sacrificed well after the date of expected delivery, and the pregnant females when their pups were 4 days old. They were killed by cervical dislocation preceded by ether anaesthesia, opened through a midline incision in the abdominal and thoracic wall and examined for gross abnormalities.

The males were all killed on the same day, 2 July 1993, after receiving test substance for 29 days. The autopsy was conducted in the same way as for the females.

On postnatal day 4, the pups were examined for grossly visible abnormalities and then subsequently discarded; stillborn and grossly malformed pups were autopsied.

Doses / concentrations

Remarks:

Doses / Concentrations:
30, 100, and 300 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

The preliminary study was carried out with 20 male and 20 female rats.

The reproduction study was carried out with 48 male and 48 female rats.

Control animals:

yes

Details on study design:

The preliminary study was carried out with 20 male and 20 female rats, selected from a group of 30 males and 30 females. At the start of the study the body weights of the males ranged from 271.3 to 335.0 g, and of the females from 180.5 to 218.8 g. The reproduction study was carried out with 48 male and 48 female rats, selected from a group of 56 males and 56 females. At the start of the study the body weights of the males ranged from 296.2 to 362.3 g, and of the females from 192.5 to 228.8 g.

The remaining rats were kept in reserve, but were not used in the study.

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

During the reproduction study no mortality occurred that could be ascribed to the administration of DETA.

Oestrous cyclicity (parental animals):

No additional information available.

Sperm parameters (parental animals):

No additional information available.

Litter observations:

The litters were evaluated on postpartum day 1 and 4 for the following parameters:
- litter size (dead and live pups)
- number of live pups
- number of dead pups
- number of stillborn pups (day 1 only)
- number of male and female pups
- number of grossly malformed pups
- litter weight
Pups that were stillborn or that died during the study were submitted to necropsy.

Postmortem examinations (parental animals):

At the end of the study, the non-pregnant females were sacrificed well after the date of expected delivery, and the pregnant females when their pups were 4 days old. They were killed by cervical dislocation preceded by ether anaesthesia, opened through a midline incision in the abdominal and thoracic wall and examined for gross abnormalities.

The males were all killed on the same day, 2 July 1993, after receiving test substance for 29 days. The autopsy was conducted in the same way as for the females.

Upon sacrifice all animals, of both the preliminary and the reproduction study, were subjected to gross autopsy under supervision of a veterinary
pathologist.

In the reproduction study, samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate buffered 4% solution of formaldehyde (F), or Bouin's fixative (B):
- ovaries, F
- testes, B
- uterus, F (after counting the implantation sites)
- epididymides, B
- seminal vesicles, F
- prostate, F
- organs and tissues showing severe lesions
Testes and epididymides were weighed prior to fixation.
The tissues for examination by light microscopy were embedded in paraffin wax, sectioned at 5 um and stained with haematoxilin and eosin. Detailed microscopic examination was conducted on all tissues and organs listed above of all animals of the control group and 300 mg DETA group.

Postmortem examinations (offspring):

On postnatal day 4, the pups were examined for grossly visible abnormalities and then subsequently discarded; stillborn and grossly malformed pups were autopsied.

Statistics:

Clinical findings of the adult rats were evaluated by Fisher's exact probability test.

Body weights and food consumption were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's Multiple Comparison test.

Fisher's exact probability test was used to evaluate the numbers of: mated and pregnant females, females with liveborn pups, females surviving delivery, females with stillborn pups or lost litters, liveborn and stillborn pups, pups lost at various stages, pups surviving 4 days, and male pups on postpartum days 1 and 4.

Precoital time, duration of gestation and litter size were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U-test, and mean pup body weights by analysis of variance followed by Dunnett's multiple comparison test.

The number of implantation sites per litter was analysed by Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U-test. For pathological changes (adults and pups) Fisher's exact probability test was used.

Reproductive indices:

Mating index = (Number of females mated/Number of females placed with males) x 100

Fertility index = (Number of females pregnant/Number of females placed with males) x 100

Fecundity index = (Number of females pregnant/Number of females mated) x 100

Gestation index = (Number of females with live pups/Number of females pregnant) x 100

Live birth index = (Number of pups born alive/Total number of pups born) x 100

Sex ratio day n = (Number of live male pups on day n/Total number of live pups on day n) x 100

Post-implantation loss ( % ) was calculated for each litter, according to the formula: (a-b)/a x 100
Where a = total number of implantation sites
b = number of pups born alive

Offspring viability indices:

Viabilit y index day 1-4 = (Number of live pups on day 4/Number of live pups on day 1 post partum) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

Diethylene triamine (DETA) was administered by gavage to male and female Wistar rats at levels of 0, 100, 300, 500 and 1000 mg/kg b.w. during 7 days to set dose levels for a reproduction/developmental toxicity screening study (OECD draft guideline no. 421). Animals in the 1000 mg DETA group showed a severe reaction to the treatment, as indicated by the mortality rate and macroscopic observations. Animals in the 500 mg DETA group showed a moderate reaction as concluded from pathological examination and decreased body weight.

During the reproduction study no mortality occurred that could be ascribed to the administration of DETA.

Data obtained through clinical observations, and macroscopic and microscopic examinations, revealed no effects of the treatment.

Mean parental body weights of both males and females in t h e 300 mg/kg b.w. DETA group were slightly decreased when compared to the control values. Body weight change of the males of the 300 mg DETA group was statistically significantl y decreased when compared t o the males from the control group from days 0-28. For the females of t h i s high dose group body weight change during the premating period (days 0-14) and during gestation (days 0-21) showed a statistically significantly decrease when compared to the control females in both periods.

Food consumption data showed a statistically significant decrease in the females of the 300 mg/kg b.w. DETA group.

Maternal performance of the females in the 100 mg and 300 mg/kg b.w. DETA groups were affected: duration of gestation was increased statistically significantly, and post-implantation loss was increased in a dose-related way. The post-implantation loss was 5.4, 3.2, 18.3, and 27.9% for receiving 0, 10, 100, and 300 mg/kg bw/day, respectively. The post-implantation loss was statistically significant at the highest dose.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Decrease body weight gain in male and females over days 0-28 (males) and gestation (females) and decreased food consumption in females at 300 mg/kg bw/day

Results: P1 (second parental generation)

Target system / organ toxicity (P1)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Details on results (F1)

No adverse effects were observed concerning the general health and the body weights of the pups.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Post-implantation loss and greater duration of gestation at 100 and 300 mg/kg bw/day.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

During the reproduction study no mortality occurred that could be ascribed to the administration of DETA. Data obtained through clinical obsevations, and macroscopic and microscopic examinations, revealed no effects of the treatment. Food consumption data showed a statistically significant decrease in the females of the 300 mg/kg bw group during the first week of the study. High dose male body weight was decreased from day 0 -28 and high dose female body weight gain was decreased during premating and during the gestation period (days 0 -21). The no-adverse-effect level for parental toxicity is 100 mg/kg bw/day.

Most mating and litter data like precoital time, mating index, fertility index, number of live and dead pups, showed no adverse effects of the treatment. Maternal performance of the females in the 100 mg/kg and 300 mg/kg bw DETA groups were affected: duration of gestation was increased in a dose-related way. Due to the relatively post-implantation loss the mean litter size was reduced in the mid and high dose group. The no-adverse-effect level for reproduction and development is 30 mg/kg bw per dy.

Pup body weights and clinical and necropsy observations did not reveal any reaction to the treatment.

Applicant's summary and conclusion

Conclusions:

On the basis of the results obtained it can be concluded that:
- Diethylenetriamine (DETA), when administered by gavage for 4 weeks, causes a light parental toxicity at levels of 300 mg/kg b.w. Consequently, the no-adverse effect level for parental toxicity as established in this study is 100 mg/kg b.w. per day.
- In view of the reproduction effects, the no-adverse effect level for reproduction and development is 30 mg/kg b.w. per day.

Executive summary:

Diethylenetriamine (DETA) was administered by gavage to male and female Wistar rats at levels of 0, 100, 300, 500 and 1000 mg/kg b.w. during 7 days to set dose levels for a reproduction/developmental toxicity screening study (OECD draft guideline no. 421). Animals in the 1000 mg DETA group showed a severe reaction to the treatment, as indicated by the mortality rate and macroscopic observations. Animals in the 500 mg DETA group showed a moderate reaction as concluded from pathological examination and decreased body weight. For the reproduction/developmental study levels of 30, 100 and 300 mg/kg b.w./day were chosen.

Diethylenetriamine was administered daily by gavage to male and female Wistar rats at levels of 30, 100 and 300 mg/kg b.w. during a 2-week premating period, and during mating and gestation up to day 4 post partum or at least during a 4-week period, to screen its effect on male and female reproductive performance and on the development of the offspring.

During the reproduction study no mortality occurred that could be ascribed to the administration of DETA. Data obtained through clinical observations, and macroscopic and microscopic examinations, revealed no effects of the treatment.

Mean parental body weights of both males and females in the 300 mg/kg b.w. DETA group were slightly decreased when compared to the control values. Body weight change of the males of the 300 mg DETA group was statistically significantly decreased when compared to the males from the control group from days 0-28. For the females of this high dose group body weight change during the premating period (days 0-14) and during gestation (days 0-21) showed a statistically significantly decrease when compared to the control females in both periods.

Food consumption data showed a statistically significant decrease in the females of the 300 mg/kg b.w. DETA group.

Maternal performance of the females in the 100 mg and 300 mg/kg b.w. DETA groups were affected: duration of gestation was increased statistically significantly, and post-implantation loss was increased in a dose-related way.

No adverse effects were observed concerning the general health and the body weights of the pups.

On the basis of the results obtained it can be concluded that:

- Diethylenetriamine (DETA), when administered by gavage for 4 weeks, causes a light parental toxicity at levels of 300 mg/kg b.w. Consequently, the no-adverse effect level for parental toxicity as established in this study is 100 mg/kg b.w. per day.

- In view of the reproduction effects, the no-adverse effect level for reproduction and development is 30 mg/kg b.w. per day.