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Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: - scientifically sound study, GLP compliant - non guideline compliant, unique study plan designed by NTP

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990
Reference Type:
secondary source
Title:
SIDS INITIAL ASSESSMENT REPORT For SIAM 18 - 1,2,3-Trichloropropane
Author:
OECD
Year:
2004
Bibliographic source:
Published OECD SIDS initial assessments of HPV chemicals: http://www.oecd.org/document/63/0,3343,en_2649_34379_1897983_1_1_1_1,00.html

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: Reproductive Assessment by Continuous Breeding (RACB) by NTP
Principles of method if other than guideline:
Task 1: dose rage finding test
Task 2: treatment of parent generation 7 d during premating and 98 d during mating (up to 5 consecutive litters)
Task 3: cross over mating of control animals with mice treated at 120 mg/Kg bw to assess the affected sex
Task 4: mating of F0 offspring (= F1) while treated at the same dose as their respective parents
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2,3-trichloropropane
EC Number:
202-486-1
EC Name:
1,2,3-trichloropropane
Cas Number:
96-18-4
Molecular formula:
C3H5Cl3
IUPAC Name:
1,2,3-trichloropropane

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS

- Strain: VAF Crl:Swiss CD-1 (ICR)BR outbred albino mice
- Source: Charles River Breeding Laboratories, Inc. (Kingston, NY, U.S.A.)
- Age at study initiation: Task 1 & 3: 8 wks, Task 2: 11 wks, Task 4: offspring of F0 generation
- Weight at study initiation: Task 1: Males: 35.0 g, Females: 25.6; Task 2 - 4: (P) Males: 36.0 g; Females: 26.4 g; (F1) Males: 33.7 g; Females: 29.4 g
- Fasting period before study: no
- Housing:
2/cage 2 per cage by sex during quarantine, Task 1 and the 1-week pre-mating period using solid bottom polycarbonate cages with stainless steel wire lids.
Task 2 animals: as breeding pairs or individually.
Bedding: SaniChip bedding (P. J. Murphy Forest Products)
- Diet (e.g. ad libitum): powdered rodent meal (NIH-07 diet) ad libitum.
- Water (e.g. ad libitum): distilled water ad libitum
- Acclimation/quarantine period: 5 - 14 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 10/14

Administration / exposure

Route of administration:
oral: capsule
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test chemical was mixed with Mazola corn oil on a weight-to-volume basis. Each dose level was independently formulated.
- Rate of preparation of solutions: Dose formulations were prepared at least every three weeks.
Dosage formulation studies over a concentration range of 0.6 to 3.0 mg/g indicated no significant loss of chemical from corn oil preparations for up to 21 days at either 5°C or room temperature in sealed vials in the dark
An aliquot of each formulation, the control, and the bulk chemical were sent to Research Triangle Institute for reference analysis during week 1 of Task 1, weeks 2, 6, 10, and 14 of Task 2, week 24 (Task 3), and weeks 20 and 28 (Task 4). (Results provided in the annex of the study report)
Corn oil was assayed for peroxides, and all batches showed less than 10 meq/kg.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not given, but corn oil is a standard for chlorinated solvents
- Concentration in vehicle:
Task1: 0, 1.25, 2.5, 5, 10 and 20 mg/mL
Tasks 2 - 4: 0, 3, 6, 12 mg/mL
- Amount of vehicle (if gavage): 10 ml/Kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: F0 generation: 98 d, F1 generation: 7 d
- Proof of pregnancy: vaginal plug referred to as day 1 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): singly
- Any other deviations from standard protocol:
Task 2 (F0 mating):
- 40 control breeding pairs and 20 breeding pairs/dose group
- treatment 7 d prior to first cohabitation and for 98 d during cohabitation,

Task 3 (F0 crossover mating trial with animals of control group and 120 mg/Kg bw group):
- animals used from Task 2 after the last Task 2 litter was weaned
- 3 groups of 20 pairs each: control males x control females, control males x high-dose females, and control females x high-dose males

Task 4: (F1 mating of animals weanlings from the last litter of Task 2):
- weaned and kept to sexual maturity (74 ± 10 days) while housed by sex two per cage (maximum) and receiving the respective treatment ( same dose groups as their parents)
- male and female from different liters cohabitated for 7 d and then housed singly until delivery
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- GC, IR, acid titration
- Dosage formulation studies over a concentration range of 0.6 to 3.0 mg/g indicated no significant loss of chemical from corn oil preparations for up to 21 days at either 5°C or room temperature in sealed vials in the dark
- An aliquot of each formulation, the control, and the bulk chemical were sent to Research Triangle Institute for reference analysis during week 1 of Task 1, weeks 2, 6, 10, and 14 of Task 2, week 24 (Task 3), and weeks 20 and 28 (Task 4). (Results provided in the annex of the study report)
Duration of treatment / exposure:
Task 1: 14 d
Task 2: for 7 d before cohabitation + 98 d during cohabitation (+ Task 3 for some animals)
Task 3: 7 d during cohabitation + time to delivery in addition to Task 2
Task 4: indirect trough the dam until weaning, directly until maturity for 74 ± 10 d + 7 days of cohabitation + time until delivery
Frequency of treatment:
once daily
Details on study schedule:
- F1 parental animals not mated until 10.5 ± 1.5 weeks after selected from the F1 litters.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Task 1: 0, 12.5, 25, 50, 100 and 200 mg/Kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
Task 2: 0, 30, 60, 120 mg/Kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Task 1: 8 per sex and dose
Task 2: 40 control breeding pairs + 20 breeding pairs per dose
Task 3: 3 groups of 20 pairs each: control males x control females, control males x high-dose females, and control females x high-dose males
Task 4: 0, 30, and 60 mg/Kg bw groups 20 breeding pairs, 120 mg/Kg bw group 10 breeding pairs do to low no of live pups in Task 2 in this dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Task 1 based on the result of the 120 d study (see chapter 7.5.1, Hazleton (1983) / key (120d study on F344 rats)); Task 2 - 4 based on the results of Task 1 ( reduced epididymis and testis weights at 125 mg/Kg bw)
- Rationale for animal assignment (if not random): stratified randomization procedure based on body weights
Positive control:
no

Examinations

Parental animals: Observations and examinations:
Task 1: clinical signs (probably daily), body weight (day1 and day 14), and water consumption (for week 1 and week 2)
Task 2: clinical signs of toxicity (probably daily), parental body weight (probably weekly, values presented for weeks 1, 2, 3, 6, 10, 14), average consumption of water weekly (values presented for weeks 2,6, 10 and 14), fertility (number producing a litter/number of breeding pairs), litters per pair, live pups per litter, proportion of pups born alive, sex of live pups and the pup body weights immediately after birth.
Task 3: same as for Task 2 + check for copulatory plug + estrual cyclicity as monitored by vaginal lavage for 12 days preceding necropsy + necropsy (organ weights, body weight, epididymal sperm motility, sperm morphology and sperm count) and histopathology (selected organs, results presented for liver, kidneys, testes, seminal vesicles, prostates, epididymides and ovaries)
Task 4: same as for Task 2 + check for copulatory plug + necropsy (organ weights, body weight, epididymal sperm motility, sperm morphology and sperm count) and histopathology (selected organs, results presented for liver, kidneys, testes, seminal vesicles, prostates, epididymides and ovaries)
Oestrous cyclicity (parental animals):
see above
Sperm parameters (parental animals):
see above
Litter observations:
see above
Postmortem examinations (parental animals):
see above
Postmortem examinations (offspring):
see above
Statistics:
Task 2: the Cochran-Armitage test is used to test for a dose-related trend in fertility
Task 3: dose groups do not represent increasing dose levels (the animals are cross-mated), therefore a chisquare test for homogeneity is used to test for an overall difference in fertility among groups. Pairwise comparisons between the control and dosed groups are made with Fisher's exact test. The proportion of live pups is defined as the number of pups born alive divided by the total number of pups produced by each pair. The sex ratio is expressed as the proportion of male pups born alive out of the total number of live pups born to each fertile pair. Dose group means for these parameters are tested for overall differences using the Kruskal-Wallis test and for ordered differences using Jonckheere's test. Pairwise comparisons of treatment group means are performed by applying the Wilcox-Mann-Whitney U test.
To remove the potential effect of the number of pups per litter on the average pup weight, an analysis of covariance is performed. The covariate used is average litter size, including live and dead pups. Least squares estimates of dose group means, adjusted for litter size, are computed and tested for overall equality using an F-test and pairwise equality using a t-test. To control for possible sex differences, these analyses are performed on males, females, and both sexes combined. An analysis of covariance is also used to adjust organ weights for total body weight. Unadjusted body and organ weights are analyzed using the Kruskal-Wallis and Wilcox-Mann- Whitney U tests. Dose-related trends are tested for by Jonckheere's test. Group means, standard deviations, and standard errors are calculated for each dose level and for each sex. Jonckheere's test is performed to obtain evidence of any increasing or decreasing trends. Significance among dose groups is calculated using Shirley's test if trend is evident or Dunn's test if no trend is evident.
Task 4: same as in Task 2 and 3
Reproductive indices:
Task 2: number of fertile breeding pairs per total breeding pairs, cumulative days to litter, average litter per pair, live pups per litter, proportion of pups born alive, sex of pups born alive, average dam weight
Task 3: average number of days to litter, mating index, pregnancy index, fertility index, live pubs per litter, proportion of pups born alive, sex of pups born alive, average dam weight
Offspring viability indices:
Task 2:live pup weight, adjusted live pup weight
Task 3:live pup weight, adjusted live pup weight

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Task 1 (pretest): all animals survived except one at the 200 mg/Kg dose level
Task 2 (F0 continuous breeding): two males and 5 females died during task 2 (dose groups not noted), at least 3 of the deaths were due to gavage trauma.
Task 3 (F0 cross-over breeding, treated animals only at 120 mg/Kg bw): no fatalities reported and no adverse clinical signs were reported
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Task 1 (pretest): all animals survived except one at the 200 mg/Kg dose level
Task 2 (F0 continuous breeding): two males and 5 females died during task 2 (dose groups not noted), at least 3 of the deaths were due to gavage trauma.
Task 3 (F0 cross-over breeding, treated animals only at 120 mg/Kg bw): no fatalities reported and no adverse clinical signs were reported
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Task 1 (pretest): only animals of the 100 and 200 mg/Kg dose groups gained weight (7 and 3 % respectively) as expected, controls and 12.5, 25 and 50 mg/Kg bw groups showed no weight gain. Historically weight gains of 6 - 8 % are to be expected.
Task 2 (F0 continuous breeding): male and female body weights of treated animals were within 10 % of the controls, but water consumption was raised in weeks 6, 10 and 14 in the 120 mg/Kg bw
Task 3 (F0 cross-over breeding, treated animals only at 120 mg/Kg bw): body weights in the 120 mg/Kg dose group were consistently lower but significant only in week 24 in females (due to variations in the pregnancy status), while no difference in water consumption was apparent
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Task 1 (pretest): only animals of the 100 and 200 mg/Kg dose groups gained weight (7 and 3 % respectively) as expected, controls and 12.5, 25 and 50 mg/Kg bw groups showed no weight gain. Historically weight gains of 6 - 8 % are to be expected.
Task 2 (F0 continuous breeding): male and female body weights of treated animals were within 10 % of the controls, but water consumption was raised in weeks 6, 10 and 14 in the 120 mg/Kg bw
Task 3 (F0 cross-over breeding, treated animals only at 120 mg/Kg bw): body weights in the 120 mg/Kg dose group were consistently lower but significant only in week 24 in females (due to variations in the pregnancy status), while no difference in water consumption was apparent
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Task 1 (pretest): not tested
Task 2 (F0 continuous breeding): tested after Task 3
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Task 1 (pretest): not applicable
Task 2 (F0 continuous breeding): no significant differences
Task 3 (F0 cross-over breeding, treated animals only at 120 mg/Kg bw): no significant differences
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Task 1 (pretest): not applicable
Task 2 (F0 continuous breeding): sperm parameters of controls and high dose (120 mg/Kg) animals analysed after Task 3
Task 3 (F0 cross-over breeding, treated animals only at 120 mg/Kg bw): Epididymal sperm motility, sperm count, and percent abnormal sperm were not affected by 1,2,3-trichloropropane treatment in the high dose (120 mg/Kg) group, low and mid dose group (30 and 60 mg/Kg bw) not tested for sperm parametres
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Task 1 (pretest): not applicable
Task 2 (F0 continuous breeding):
- with increasing dose and duration of exposure, fertility (pregnancy per breeding pair) is reduced at both the middle and high doses (60 and 120 mg/Kg bw).
- also litters per fertile pairs are reduced dose dependently, reaching significance at the high dose (120 mg/Kg bw)
- no of live pups per litter are reduced dose dependently, reaching significance for litters 2- 5 at the high dose (120 mg/Kg bw) and at the 5 th litter in the mid dose (60 mg/Kg bw). Interestingly the proportion of pups born alive was not affected by the treatment
- a dose-related decrease in the proportion of males pups born alive in the 5th litter was noted
- cumulative days to litter for the 3rd litter at 60 mg/kg and the 4th and 5th litter at 120 mg/kg were longer than the control values
Task 3 (F0 cross-over breeding, treated animals only at 120 mg/Kg bw):
- mating index (number of pairs with copulatory plugs divided by the number mated) and fertility index (number of fertile pairs divided by number copulatory plug positive) were NOT different among the groups
- treated females delivered fewer live pups, the males of which were significantly lighter than controls.
- there was a trend to fewer males in the litters of treated females.
none of these trends reached significance, therefore a it cannot be singled out which sex is mainly affected

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive performance, on sex ratio, on pup weight and reproductive organ weights

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Task 4 (F1 breeding): parental animals of Task 4 are the offspring of the last mating in Task 2 and had already raised body weights as pups in the 60 and 120 mg/Kg bw groups. This tendency persisted and was significant until their sacrifice at the end of Task 4.
Mortality / viability:
no mortality observed
Description (incidence and severity):
Task 4 (F1 breeding): no fatalities reported and no adverse clinical signs were reported
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Task 4 (F1 breeding): Live male and female pup weights (offspring of Task 2) were significantly higher in the 120 mg/kg group while in the 60 mg/kg group only a trend for higher values was noted (possibly due to lower litters sizes)
Sexual maturation:
no effects observed
Description (incidence and severity):
no effects
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Task 4 (F1 breeding):
- male reproductive organs normal in all treatment groups.
- male absolute and relative liver and kidney weights raised at 60 and 120 mg/Kg bw, relative testis weights reduced only at 60 mg/Kg bw
- female absolute and relative liver weights, absolute kidney weights raised at 60 and 120 mg/Kg bw, and relative ovary weights reduced at 60 and 120 mg/Kg bw, absolute ovary weight reduced only at 120 mg/Kg bw
Gross pathological findings:
no effects observed
Description (incidence and severity):
Task 4 (F1 breeding): no significant findings
Histopathological findings:
no effects observed
Description (incidence and severity):
Task 4 (F1 breeding): no significant findings
Other effects:
effects observed, treatment-related
Description (incidence and severity):
OTHER FINDINGS (OFFSPRING)
REPRODUCTIVE FUNCTION: ESTROUS CYCLE (OFFSPRING)
Task 4 (F1 breeding): average estrous cycle length for treated females was longer than the control value at all dose levels

REPRODUCTIVE FUNCTION: SPERM MEASURES (OFFSPRING)
Task 4 (F1 breeding): no significant findings

REPRODUCTIVE PERFORMANCE (OFFSPRING)
Task 4 (F1 breeding):
- a significant decrease was noted for mating and fertility indices in the 120 mg/kg group
- number of live pups in the 120 mg/kg group was decreased but the difference was not significant

Effect levels (F1)

Dose descriptor:
LOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: average estrous cycle lengths were significantly increased for all 1,2,3-trichloropropane-exposed females of the F1 generation at all tested doses

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The reproductive toxicity of 1,2,3-trichloropropane was tested in a 2 generation study with unique test design (continuous breeding protocol) where CD-1 Swiss mice were treated via gavage (vehicle = corn oil). In the parent generation adverse effects on the reproductive performance, on sex ratio, on pup weight and on reproductive organ weights were seen at 60 and 120 mg/Kg bw, at which slight signs of parental toxicity were noted (moderately raised liver and kidney weights). In the offspring these effects were confirmed but only for the 120 mg/Kg group, except for effects on estrus cyclicity that were seen down to 30 mg/Kg bw .
Executive summary:

In the present study (Gulati 1990) the reproductive toxicity of 1,2,3-trichloropropane was tested in a 2 generation study with unique test design (continuous breeding protocol) where CD-1 Swiss mice were treated via gavage with 0, 30, 60 and 120 mg/Kg bw in corn oil.

In the parent generation adverse effects on the reproductive performance, on sex ratio, on pup weight and on reproductive organ weights were seen at 60 and 120 mg/Kg bw, at which slight signs of parental toxicity were noted (moderately raised liver and kidney weights). The effects on reproductive performance was both time and dose dependent. In the offspring these effects were confirmed but only for the 120 mg/Kg group, except for effects on estrus cyclicity that were seen down to 30 mg/Kg bw. The induced toxicity was regarded to slight to explain the marked adverse effects in both generations. The cross mating experiment (Task 3) failed to clearly identify the affected sex, though the data suggests a slightly greater reproductive toxicity for females.

Based on these results 1,2,3 -trichloropropane is deemed a reproductive toxicant. Due to the limited set of investigations, a clear NOAEL is difficult to derive. Following a conservative approach based on the effects on estrous cyclicity in the F1 generation a LOAEL of 30 mg/Kg bw is deduced.

This assessment is in-line with the evaluation of the Concise International Chemical Assessment Document 56 on 1,2,3-TRICHLOROPROPANE (International Program on Chemical Safety (IPCS) 2003, http://www.inchem.org/documents/cicads/cicads/cicad56.htm)

In the study 4 different Tasks were performed:

Task 1: dose rage finding test

Task 2: treatment of parent generation 7 d during premating and 98 d during mating (up to 5 consecutive litters)

Task 3: cross over mating of control animals with mice treated at 120 mg/Kg bw to assess the affected sex

Task 4: mating of F0 offspring (= F1) while treated at the same dose as their respective parents

The following investigations were performed in the respective task:

Task 1 (pretest): 8 per sex and dose, clinical signs (probably daily), body weight (day1 and day 14), and water consumption (for week 1 and week 2).

Task 2 (F0 continuous breeding): 40 control breeding pairs + 20 breeding pairs per dose, clinical signs of toxicity (probably daily), parental body weight (probably weekly, values presented for weeks 1, 2, 3, 6, 10, 14), average consumption of water weekly (values presented for weeks 2,6, 10 and 14), fertility (number producing a litter/number of breeding pairs), litters per pair, live pups per litter, proportion of pups born alive, sex of live pups and the pup body weights immediately after birth.

Task 3 (F0 cross-over breeding, treated animals only at 120 mg/Kg bw): 3 groups of 20 pairs each: control males x control females, control males x high-dose females, and control females x high-dose males, same as for Task 2 + check for copulatory plug + estrual cyclicity as monitored by vaginal lavage for 12 days preceding necropsy + necropsy (organ weights, body weight, epididymal sperm motility, sperm morphology and sperm count) and histopathology (selected organs, results presented for liver, kidneys, testes, seminal vesicles, prostates, epididymides and ovaries).

Task 4 (F1 breeding): 0, 30, and 60 mg/Kg bw groups 20 breeding pairs, 120 mg/Kg bw group 10 breeding pairs do to low no of live pups in Task 2 in this dose group, same as for Task 2 + check for copulatory plug + necropsy (organ weights, body weight, epididymal sperm motility, sperm morphology and sperm count) and histopathology (selected organs, results presented for liver, kidneys, testes, seminal vesicles, prostates, epididymides and ovaries).