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Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: 90-day toxicity study
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted under GLP and it was done according to valid methods for repeated dose toxicity. The study is reliable and relevant, however it is not fully adequate for classification.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: EU Method B.27 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD 408
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Wistar; Hoe: WISKf(SPF71)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG, Hattersheim-Kastengrund (Germany), SPF-Zucht,
- Age at study initiation: Males ca. 4 weeks; females ca. 5 weeks
- Weight at study initiation: Average males 79.8 g; average females 85.4 g
- Housing: Draft mesh cages (Type 3), in groups until 2 animals
- Diet: Rattendiät Altromin 1321 by Altromin GmbH Lage/Lippe (Germany) ad libitum
- Water: Tap water in plastic bottles, ad libitum
- Acclimation period: At least 5 days before study

ENVIRONMENTAL CONDITIONS
Fully airconditioned rooms
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Extraction of the active ingredient from the spiked diet using methanol (Chromasolv R). Concentration and dilution in methanol
(Chromasolv R). Determination of the active ingredient using gaschromatography with a nitrogen selective detector (PND).
(cf report Dr. Thier dd 1984-07-12 / encluded in the study report)
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 2, 10, 50 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In an earlier 90 days oral repeated dose study doses of 12.5 and 50 mg/kg bw/day were administered. These doses were too high to determine a no effect level. Purpose of this experiment was to determine a no effect level.
- Rationale for selecting satellite groups: first 5 animals of each group were killed at the end of testing; the other 5 were killed after 29 days recovery period.
- Post-exposure recovery period in satellite groups:29 days
Parental animals: Observations and examinations:
Monday - Friday 2x daily, on weekends 1x daily: behaviour /health state
1x weekly: neurological disorders, opacities of eyes, injuries of the mouth's mucous membrane, disturbed toothgrowth
2x weekly, during acclimation, study and recovery: body weight
2x weekly during study: feed used; 1x weekly water used
end of study or end of recovery period: haematological parameters in the blood
after sacrifice: clinical-chemical parameters in the serum
night between 88./89. study day, 16 h urine: urinanalysis (animals unfeed, unwatered)
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
- Sacrifice by i.p. injection of 50 mg Nembutal / kg bw / de-bleeding by opening the vena cava cranialis
- gross assessment according to Hoechst Sektionsordnung I, Prof. K/G dd 1982-02-04
- relative organ weights (% of bw)
HISTOPATHOLOGY: Yes
- histological assessment of fixated organ or preparations according to Hoechst Sektionsordnung I, Prof. K/G dd 1982-02-04: heart, lungs, liver, kidneys, spleen, stomach, jejunum, colon, bladder, testes, epidiymis, prostate, seminal vesicle, ovaries, uterus,thyroid, pancreas, adrenals, thymus, pituitary gland, brain, eyes with N. opticus, bone marrow (femur marrow)
Statistics:
Statistical evaluation of following measured values, using probability level 0.05:
- body weight at observation times
- body weight gain during intervals
- haematological parameters (except differential blood count)
- clinical chemical parameters
- urinanalysis (pH, density)
- absolute/ relative organ weights
Hoechst evaluation software used according to SOP Dr. Passing, Department of Practical Mathematics.

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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed
Reproductive function: oestrous cycle:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY: no effects, no mortalities during dosing & recovery period.
BODY WEIGHT AND WEIGHT GAIN: decreased in males & femaels in highest dose from week 2/3 onwards, respectively; reversible in recovery period.
FOOD CONSUMPTION AND COMPOUND INTAKE: decreased in males & females during first 30 dyas. No effects in recovery period.
FOOD EFFICIENCY: no data
WATER CONSUMPTION: no effects during dosing & recovery period.
OPHTHALMOSCOPIC EXAMINATION: no effects during dosing & recovery period.
HAEMATOLOGY: increased white blood cells at highest dose; reversible in recovery period.
CLINICAL CHEMISTRY: no effects during dosing & recovery period.
URINALYSIS: no effects during dosing & recovery period.
NEUROBEHAVIOUR: no effects during dosing & recovery period.
ORGAN WEIGHTS: male rats, highest dose: reduced testicular weights; still observed in the recovery group - decreased liver weight in females, highest dose; reversible in recovery period however decreased liver weight in males after recovery period.
GROSS PATHOLOGY: male rats, highest dose: smaller testes, reversible in the recovery group
HISTOPATHOLOGY: male rats, highest dose: depression - standstill of spermiogenesis and proliferation of Leydig cells and other interstitial cells in testes; absence of mature & immature sperms instead only loose connective tissue visible in epidydimides; in the recovery group starting to complete regeneration was observed.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg diet
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
other: Generation not specified (migrated information)
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
other: Generation not specified (migrated information)
Reproductive effects observed:
not specified
Conclusions:
After the highest dose, male rats had smaller testes, disturbed spermiogenesis and proliferation of the Leydig cells.
After a 29 recovery period beginning or complete regeneration of testes tubuli were observed.

Chloroacetamide in this 90 day oral repeated dose study in rats caused a decreased body weight gain and increased leucocyte count in the high dose male and female rats. The high dose males had smaller testes, depression- standstill of the spermiogenesis and proliferation of Leydig cells. After a 29 day recovery period, there were signs of beginning or ended regeneration of the testicular tubuli.
The NOAEL for Chloroacetamide in this study was determined to be 10 mg/kg bw/day.
Executive summary:

In a 90 day oral repeated dose study in Wistar rats were given doses of 0, 20, 100 and 500 mg Chloroacetamide test substance/kg feed /day. Each group consisted of 10 male and 10 female animals, of which the first 5 animals of each group, were killed at the end of the 90 day, whereas the other 5 were killed after a 29 day recovery period. In the high dose (500 mg/kg feed) male and female animals showed reduced body weight gain and food consumption and increased leucocytes in both sexes, decreased liver weights in females, and smaller testes, reduced testicular weights, proliferation of Leydig/other interstitial cells and depression - standstill of spermiogenesis in males.Re-onset in the recovery group with partial to complete regeneration of testes was observed in males, therefore this was to be designated as reversible. Further liver weights were reversed in females and slightly decreased in males in the hightest dose recovery groups. The NOAEL was determined to be 100 mg/kg feed, which was reported to correspond with 10 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
High (Klimisch 2)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information
Following studies were available demonstrating a reversible effect on male reproduction organs and reproductive capacity.

In a 90 day oral repeated dose study, Wistar rats were given doses of 0, 20, 100 and 500 mg Chloroacetamide test substance/kg feed /day (Hoechst AG, 1985). Each group consisted of 10 male and 10 female animals, of which the first 5 animals of each group, were killed at the end of the 90 day, whereas the other 5 were killed after a 29 day recovery period. In the high dose (500 mg/kg feed) male and female animals showed reduced body weight gain and food consumption and increased leucocytes in both sexes, decreased liver weights in females, and smaller testes, reduced testicular weights, proliferation of Leydig/other interstitial cells and depression - standstill of spermiogenesis in males. Re-onset in the recovery group with partial to complete regeneration of testes was observed in males, therefore this was to be designated as reversible. Further liver weights were reversed in females and slightly decreased in males in the highest dose recovery groups. in the females, follicle maturation was found to be normal. The NOAEL was determined to be 100 mg/kg feed, which was reported to correspond with 10 mg/kg bw/day. In a second 90 day study in rats ( Leuschner, 1970), testicular damage observed at 50 mg/kg bw was also seen at 12.5 mg/ kg bw, though less pronounced at the latter dose.

A dominant lethal test was performed in which male NMRI-mice received intraperitoneal administration of Konservierungsmittel CA 24 (containing 70% Chloracetamide) at 114 mg/kg bw (group 1) and 123 mg/kg bw (group 2) and were subsequently mated with untreated females for several weeks. At autopsy of the mated females, following findings were observed at 123 mg/kg bw in comparison to the control group:
1. Decreased number of implantations after 1, 2 and 3 weeks, highly significant decrease after 1 and 3 weeks
2. Decreased fertility index during the first and second week
3. Slightly significant decreased number of fetuses after 1 and 2 weeks, highly significant decrease after 3 weeks.
The described findings most probably were an expression of a toxic effect in the males during the first 3 weeks of testing, which proved to decline from the fourth week. A marked inhibitory effect on fertility of the treated males was observed in the first 3 weeks of testing (IBR, 1979c).

In conclusion, possible risk of impaired fertility impact with (partial) recovery was seen in two studies.

As classification was proposed, no further testing is proposed.


Short description of key information:
In a 90-day toxicity study in rats, a reversible effect on male reproductive organs was observed at 500 mg test substance/kg feed, a dose level showing also parental toxicity; no effects were observed at the lower dose level of 100 mg/kg feed, corresponding with 10 mg/kg body weight/day. The effect was partially reversible after 1 month recovery. In a dominant-lethal assay in mice, inhibitory effects on fertility of the treated males was observed, which were considered to be related to toxic effects in the male reproductive system during the first 3 weeks of testing.

Justification for selection of Effect on fertility via oral route:
The study was conducted under GLP and it was done according to valid methods for repeated dose toxicity.

Effects on developmental toxicity

Description of key information
Studies with acetamide and chloracetamide given systemically in pregnant rats were available as weight of evidence. Both subcutaeneous and intraperitoneal dosing once or twice during pregnancy at dose levels approximating half the LD50 or more did not lead to malformations of the fetuses. 
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Studies with acetamide and chloracetamide given by subcutaneous and intraperitoneal administration in pregnant rats were available as weight of evidence.

Acetamide, given by single subcutaneous application up to 2000 mg/kg bw (80% of LD50) on day 13 of gestation in CD and BD/X rats, did not demonstrate fetal abnormalities on day 14 -17 (24 -96h) after caesarian section (von Kreybig et al., 1968). Chloroacetamide, given by single subcutaneous application at 70 mg/kg bw (71% of LD50) on day 13 of gestation in CD and BD/X rats, did not demonstrate fetal abnormalities on day 14 -17 (24 -96h) after caesarian section (von Kreybig et al., 1969). The dose proved toxic and resulted in postnatal death in 50% of the pups. There were no cases of malformations. Development was normal in the survivors.

When Chloroacetamide was administered intraperitoneally (LD50 intraperitoneal 50 mg/kg bw) to pregnant Long-Evans rats at 20 mg/kg body weight either as a single dose on day 7 or on days 11 and 12 of gestation and caesarean section was performed in both groups on day 21 of gestation, assessment of implantations, placental weights, foetal weights and foetal malformations revealed no cases of embryotoxicity or teratogenicity. Moreover,the dams showed no signs of toxicity (Thiersch, 1967).

Justification for classification or non-classification

Chloroacetamide was classified by the Federal Republic of Germany according the EU labelling regulations Commision Directive 93/21/EEC as HARMFUL as Category 3 with risk phrase R 62 ‘POSSIBLE RISK OF IMPAIRED FERTILITY'. According to CLP regulation (No. 1272/2008 of 16 December 2008), Category 2 classification is proposed with Signal word 'WARNING' and hazard statement H361 ‘SUSPECTED OF DAMAGING FERTILITY OR THE UNBORN CHILD<REVERSIBLE MALE REPRODUCTIVE ORGAN CHANGES > <ORAL ROUTE>’.