Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
multi-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
before 1970
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented study. Short comings are: small animal number, no definite data on actual intake of L-Cysteine hydrochloride monohydrate, no purity and no impurities given for the test substance. In results: only means given; no min/max no standard deviation given (thus, results are not presented in very detail). It is important to mention that the substance was applied within the bread baking procedure and heated up to 227°C for 1/2 hour. This may have influenced the stability of the test substance especially as there are comments declaring a decomposition temperature of 175-177.8 °C for L-Cysteine hydrochloride. Nevertheless the documentation is complete enough, so that the influence of the baking process could be evaluated by an additional test if needed. Therefore the study can be jugded as a valid screening test for the evaluation of toxic effects on fertility.

Data source

Reference
Reference Type:
publication
Title:
Use of L-Cysteine in Bread Baking - Results of a Multi-generation Feeding Experiment with Breeding Rats
Author:
Frape DL, Wilkinson J, Chubb LG, Buchanan AM, Coppock JBM
Year:
1971
Bibliographic source:
J. Sci. Fd Agric. 22: 65-68

Materials and methods

Principles of method if other than guideline:
L-Cysteine hydrochloride monohydrate was added to flour at levels of 0, 35, 350 and 3500 ppm. Bread was baked from this flour, dried and milled to a powder, which was added to the rats diet.
Starting with 4 pregnant rats in the control and the highest dosage group the mothers and all pups were slaughtered except for 2 male and 3 female pups selected at random from each litter, which were retained for breeding.
This procedure was continued in the second and third generations, but at the weaning of the fourth generation only 24 males and 48 females were retained for breeding, i.e. 12 males and 24 females on each diet. The same procedure as before was adopted for the sixth generation, using all four dosages, and during the breeding of the seventh generation the experiment was terminated.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): L-Cysteine Hydrochloride Monohydrate
- Molecular formula (if other than submission substance): C3H8O2NSClxH2O
- Molecular weight (if other than submission substance): 175.64
- Analytical purity: not reported
- Impurities (identity and concentrations): no data
- Stability under test conditions: not known. However, it is important to mention that the substance was applied within the bread baking procedure and heated up to 227°C for 1/2 hour. This may have influenced the stability of the test substance. In a Material Safety Data Sheet from Fisher Scientific (ACC#06129) the decomposition temperature is given with 175-177.8 °C.
IN ACCORDANCE WITH ARTICLE 2(7)(b) and Annex V (6), REACH the L-Cysteine hydrochloride is registered (hydrate free form). L-Cysteine, hydrochloride, monohydrate (CAS 7048-04-6) is manufactured and imported and was used for testing.

Test animals

Species:
rat
Strain:
other: C.D. Norwegian Hooded of an inbred Middle Aston strain
Sex:
female
Details on test animals and environmental conditions:
Starting with pregnant C.D. Norwegian Hooded rats of an inbred Middle Aston strain.

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
The substance was added to flour in concentrations of 0, 35, 350 and 3500 ppm. With this flour bread was baked. After the baking the bred was sliced, freeze-dried, crushed and milled. This powder was mixed to a diet. The diets contained about 77% of bread crumbs on dry weight
No feed intake is reported. An animal weight of 100 g and a feed intake of 10 g diet per animal and day is used for estimation of the doses. 77% of milled bread was added to the diet. This resulted in an estimated daily intake of L-Cysteine of 0, 2.7, 27 and 270 mg/kg bw..
Details on mating procedure:
For the first generation pregnant rats were used. No data on mating procedure available. 11 weeks after birth the next generation females were mated with males from the same litter. During the breeding of the seventh generation the experiment was terminated.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 generations
Frequency of treatment:
daily
Details on study schedule:
L-Cysteine hydrochloride monohydrate was added to flour in concentrations of 0, 35, 350 and 3500 ppm. With this flour bread was baked. After the baking a powder was produced and mixed to a diet. The test article intake is estimated: An animal weight of 100 g and a daily feed intake of 10 g diet per animal and day is presumed. 77% of milled bread was added to this diet. This resulted in an estimated daily intake of L-Cysteine hydrochoride monohydrate of 0, 2.7, 27 and 270 mg/kg bw..
For the first generation pregnant rats were used. Starting with 4 pregnant rats in the control and the highest dosage group the mothers and all pups were slaughtered after weaning except for 2 male and 3 female pups selected at random from each litter, which were retained for breeding.
11 weeks after birth the next generation females were mated with males from the same litter.
This procedure was continued in the second and third generations, but at the weaning of the fourth generation only 24 males and 48 females were retained for breeding, i.e. 12 males and 24 females on each diet. The same prodceure as before was adopted for the sixth generation, using all 4 dosages, and during the breeding of the seventh generation the experiment was terminated.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 ppm
Basis:
other: flour weight
Remarks:
Doses / Concentrations:
35 ppm
Basis:
other: flour weight
Remarks:
Doses / Concentrations:
350 ppm
Basis:
other: flour weight
Remarks:
Doses / Concentrations:
3500 ppm
Basis:
other: flour weight
No. of animals per sex per dose:
4
Control animals:
yes
Details on study design:
Estimation of the actual intake of L-Cysteine hydrochloride monohydrate:
feed intake: animal weight: 100g, 10 g diet per animal and day. 77% of milled bread was added to the diet. This resulted in an estimated daily intake of L-Cysteine of 0, 0.27, 2.7 and 27 mg per animal.
Positive control:
Controls: yes
Solvent control: not applicable
Positive control: no

Examinations

Postmortem examinations (parental animals):
examinations for gross lesions
Postmortem examinations (offspring):
examinations for gross lesions
Reproductive indices:
Number born, litter weight at birth, numbers weaned and litter weight at weaning, carcass, liver and kidney weights of all slaughtered aninmals, post mortem examination for gross lesions.
In the 0 and 3500 ppm groups the principal characteristics of the response to the treatment were measured: rate of change per generation in litter size and weight at birth and at weaning, and the rate of change in the size of the carcasses, livers and kidneys of both adults and weanling rats.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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 performance:
no effects observed

Details on results (P0)

Breeding rats of the fifth generation at about 150 days of age and receiving the diet with the highest L-cysteine hydrochloride monohydrate treatment (3500 ppm) were examined histopathologically. They showed no lesions atypical in normal rats of that age.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

As results only means given; no min/max no standard deviation given.

No treatment related effects were observed (authors: "no obvious adverse effect").

Applicant's summary and conclusion

Conclusions:
L-Cysteine hydrochloride monohydrate was added to flour. With this flour bread was baked and from this bread a powder was produced and mixed to the diet. A daily intake of 0, 2.7, 27 and 270 mg/kg bw. was estimated..
Starting with 4 pregnant rats in the control and the highest dosage group a study over 7 generations was conducted.

Although quite well documented the study has short comings as there are: small animal number, no definite data on actual intake of L-cysteine hydrochloride monohydrate, no purity and no impurities given for the test substance and in results: only means, no min/max no standard deviation are given. Nevertheless the study can be jugded as a valid screening test for the evaluation of the reproduction toxicity.

Up to and including dosages of 270 mg/kg bw. no adverse effects on fertility occurred in the rats during the experiment even at the high level of L-cysteine hydrochloride monohydrate treatment.
Executive summary:

L-Cysteine hydrochloride monohydrate was added to flour in concentrations of 0, 35, 350 and 3500 ppm. With this flour bread was baked. After the baking the bred was sliced, freeze-dried, crushed and milled. This powder was mixed to a diet. The diets contained about 77% of bread crumbs on dry weight.

Estimation of doses: An animal weight of 100 g and a daily feed intake of 10 g diet per animal and day is estimated. 77% of milled bread was added to this diet. This resulted in an estimated daily intake of L-cysteine hydrochloride monohydrate of 0, 2.7, 27 and 270 mg/kg bw..

For the first generation pregnant rats were used.

Starting with 4 pregnant rats in the control and the highest dosage group the mothers and all pups were slaughtered after weaning except for 2 male and 3 female pups selected at random from each litter, which were retained for breeding.

11 weeks after birth the next generation females were mated with males from the same litter.

This procedure was continued in the second and third generations, but at the weaning of the fourth generation 24 males and 48 females were retained for breeding, i.e. 12 males and 24 females on each diet. The same prodceure as before was adopted for the sixth generation, using all 4 dosages, and during the breeding of the seventh generation the experiment was terminated.

Recorded are number born, litter weight at birth, numbers weaned and litter weight at weaning, carcass, liver and kidney weights of all slaughtered aninmals, post mortem examination for gross lesions. In the 0 and 3500 ppm groups the principal characteristics of the response to the treatment were measured: rate of change per generation in litter size and weight at birth and at weaning, and the rate of change in the size of the carcasses, livers and kidneys of both adults and weanling rats.

Breeding rats of the fifth generation at about 150 days of age and receiving the diet with the highest

L-cysteine hydrochloride monohydrate

treatment (3500 ppm) were examined histopathologically. They showed no lesions atypical in normal rats of that age.

No adverse effects on fertility occurred in the rats during the experiment even at the high level of L-cysteine hydrochloride monohydrate treatment.

It is important to mention that the substance was applied within the bread baking procedure and heated up to 227°C for 1/2 hour. This may have influenced the stability of the test substance especially as there are comments declaring a decomposition temperature of 175-177.8 °C for l-cysteine hydrochloride. Nevertheless the documentation is complete enough, so that the influence of the baking process could be evaluated by an additional test if needed.