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Toxicological information

Toxicity to reproduction

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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:
06 August 2014 to 14 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Epofloc L-1R
IUPAC Name:
Epofloc L-1R
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
- Name of test material (as cited in study report): Reaction products of N-(2-aminoethyl)-N'-{2-[(2-aminoethyl)amino]ethyl}ethane-1,2-diamine,N-(2-aminoethyl)-N'-[2-(piperazin-1-yl)ethyl]ethane-1,2-diamine,N-{2-[4-(2-aminoethyl)piperazin-1-yl]ethyl}ethane-1,2-diamine,N,N,N'-tris(2-aminoethyl)ethane-1,2-diamine, carbon disulphide and sodium hydroxide. EC939-782-1
- Substance type: Heavy metal scavenger
- Physical state: Liquid
- Analytical purity: 40.5% solid content determination
- Purity test date: 22 November 2013
- Lot/batch No.: 311045D
- Expiration date of the lot/batch: 22 Novenber 2014
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature, in tightly closed container in well ventilated area.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Commercial laboratory animal supplier (Animal Breeding Facility, Jai Research Foundation)
- Age at study initiation: (P) 10 - 11 wks;
- Fasting period before study: no
- Housing: Individually in solid floor polypropylene rat cages (Dimensions: 41 x 28.2 x 18 cm), except during the mating period where 1 male and 1 female were housed per cage
- Diet: Standard rodent pellet ad libitum
- Water: filtered through reverse osmosis water filtration system, ad libitum.
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 to 24°C
- Humidity: 66 to 68%
- Air changes (per hr): 19-20
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 18 August 2014 (treatment) to 18 November 2014 (experimental completion)

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

TEST FORMULATIONS (frequency): Test formulations were freshly prepared every day prior to dosing and used within 2 hours

VEHICLE
- Concentration in vehicle: 10 ml/kg bw

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until copulation occurred
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Accuracy of dose formulation was determined by weighing and was calculated as the dry weight of test material in the test sample. Study formulations were analysed on three occasions and all of the results except one were > 20 % above target.
Duration of treatment / exposure:
Dosing of both sexes was initiated 2 weeks prior to mating and continued during the mating period. After mating, the male rats were further dosed up to and including the day before scheduled sacrifice, until approximately 80% of the females had delivered. Female rats were further dosed during pregnancy and at least up to post-partum day 4. Rats from the recovery groups were kept for 16 days after the first scheduled sacrifice of dams, without treatment, to detect delayed occurrence, or persistence, or recovery from toxic effects, if any.
Frequency of treatment:
Once, daily, 7 days a week.
Details on study schedule:
Not applicable
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
30 animals: 15 male and 15 female (plus 5 male and 5 female (non-mated) in the control and high dose recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on dose range finding study (JRF Study No. 615-1-01-8067) at the recommendation of the sponsor.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No] were included in report

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Male body weights were recorded on the first day of dosing and at weekly intervals. Female bodyweights were recorded on the first day of dosing and at weekly intervals during the pre-mating and mating period, and on gestation days 0, 7, 14, 20 and day 0 and 4 post parturition. Body weight was recorded on the day of fasting for respective animals and for all animals at sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Male food consumption was measured weekly during pre-mating and post-mating periods. Female food consumption was recorded on the same day as bodyweight recording during the pre-mating and gestation period, and on day 0 and 4 post parturition.
Oestrous cyclicity (parental animals):
Oestrous cycle examination undertaken but no data available.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: Testicular histopathological examination was conducted with special emphasis on stages of spermatogenesis and histopathology interstitial testicular cell morphology.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 post-partum: yes
- Female rats along with pups which were not selected for further evaluation were killed (post-partum day 4) and discarded

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number and sex of pups, stillbirths, dead pups, undersized pups and the presence of gross anomalies. Body weight was recorded on lactation days 0 and 4

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups which died prior to scheduled necropsy.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed after approximately 80% of females had delivered.
- Maternal animals: All surviving animals were sacrificed on post-partum day 5.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera and organ wieghts.

HISTOPATHOLOGY
- Conducted in all tissues from control and high dose animals (testes, epididymides and ovaries were subject to special examination, and kidneys were examined in all male groups)

Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals.
- These animals were subjected to postmortem examinations. (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:
Bartlett's test for heterogeneity of variance was followed by ANOVA and Dunnett's or t test. Categorical variables were analysed using Chi squared test.
Reproductive indices:
Pre-implantation loss, post-implantation loss, mating index, fertility index and gestation index.
Offspring viability indices:
Live birth index and viability index (on day 4 post-partum)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
significant decrease at 1000 mg/kg bw/day
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
significant decrease at 1000 mg/kg bw/day
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Inflammatory changes in kidneys of males at 1000 mg/kg bw/day
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: Analysis of dose formulations indicated 20 - 30 % over target levels, implying that test groups received at least a 20% dose overage

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Increased post implantation loss at 1000 mg/kg bw/day

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): no toxic effects.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS); significant decrease in bodyweight and feed consumption

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): no effect

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): no data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Increased post implantation loss at 1000 mg/kg bw/day

ORGAN WEIGHTS (PARENTAL ANIMALS): no data

GROSS PATHOLOGY (PARENTAL ANIMALS): no effect

HISTOPATHOLOGY (PARENTAL ANIMALS): Microscopic examination of the kidneys revealed basophilic/regenerating tubules in 5/5 male animals at 1000 mg/kg bw /day (high dose) compared to no observations in the control, low and mid dose group animals. At 1000 mg/kg bw /day lesions persisted even after a recovery period with an almost similar incidence and severity.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Increased post natal loss at 300 and 1000 mg/kg bw/day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant decrease in bodyweight at 100, 300 and 1000 mg/kg bw/day.
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified

Details on results (F1)

VIABILITY (OFFSPRING): increase in post natal loss at 300 and 1000 mg/kg bw/day

CLINICAL SIGNS (OFFSPRING): no effect

BODY WEIGHT (OFFSPRING): decrease in bodyweight relative to control at 100, 300, and 1000 mg/kg bw/day

SEXUAL MATURATION (OFFSPRING): no effect

ORGAN WEIGHTS (OFFSPRING): no effect

GROSS PATHOLOGY (OFFSPRING): no effect

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Administration of the test substance during the treatment period produced a significant decrease in bodyweight, bodyweight change and feed consumption, increased post implantation loss, decreased live birth/survival index and inflammatory changes in the kidneys at 1000 mg/kg bw/day. The effects upon bodyweight and kidney were still apparent in recovery group animals two weeks after cessation of treatment at 1000 mg/kg bw/day.
Administration of the test item at 100 or 300 mg/kg bw/day failed to produce toxic effects in parent animals.
There was a decrease in pup viability at 300 or 1000 mg/kg bw/day and a decrease in pup body weight at 100, 300 or 1000 mg/kg bw/day.

Hence, it is concluded that the NOAEL is 300 mg/kg bw/day for parental animals and for pups the NOAEL is 100 mg/kg bw/day.