3-amino-2-chlor-6-methylphenol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05.01. - 26.01.2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

The 50% formulation used for the first induction of the additional animals was prepared within 5 h prior to treatment. An effect on the stability of the substance was not to be expected. The protocol deviation did not adversely affect the study integrity.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

80419-48-3

Test material form:

solid

Details on test material:

beige powder

Specific details on test material used for the study:

Analogy
CAS 80419-48-3
2-Chlor-3-amino-6-methylphenol x HCl

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

30 females, nulliparous and non-pregnant
Age: approx. 11 weeks
Body weight: variation was within +/- 20% of the sex mean
Conditions:
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 18.9 - 22.2°C), a relative humidity of 30-70% (actual range: 33 - 72%) and 12 hours artificial fluorescent light and 12 hours darkness per day. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity.
Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
Accommodation:
Individual housing in labelled Macrolon cages (type I; height 12.5 cm) containing purified sawdust as bedding material (Woody-Clean type 3/4; Tecnilab-BMI BV, Someren, The Netherlands). Certificates of analysis were examined and then retained in the NOTOX archives. The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions. Animals were group housed in polycarbonate cages (Macrolon II type; height 15 cm) during the acclimatisation period.
Diet:
Free access to standard pelleted laboratory animal diet (from Altrom in (code VRF 1 ), Lage, Germany).
Water:
Free access to tap-water.

Study design: in vivo (non-LLNA)

Details on study design:

Preliminary irritation study
A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (grade 3) at the highest.
A series of four test substance concentrations were tested, the highest concentration being the maximum concentration that could technically be applied. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 25%, 10%, 5%, 2.5%, 1 % and if needed further lower concentrations using the same steps. The test system, procedures and techniques were identical to those used during days 1 to 3 of the main study unless otherwise specified.
Four young adult animals were selected (5-14 weeks old). Each animal was treated with one concentration on two consecutive days. Approximately 4 hours after the last exposure, the skin was cleaned of residual test substance with water and the irritation was assessed. No necropsy was performed and no bodyweights were determined after termination.

Main study
lnitially, four groups of five animals were treated with the vehicle and three test substance concentrations respectively. Based on the results, two additional groups were treated with the vehicle and the highest test substance concentration.

Study design: in vivo (LLNA)

Vehicle:

other: ethanol:water (7:3 v/v)

Concentration:

5%, 25% and 50%

No. of animals per dose:

5

Details on study design:

Test substance concentrations selected for the main study were based on the results of a preliminary study.
In the main study, three groups of five experimental animals were epidermally exposed to a 5%, 25% and 50% concentration respectively on three consecutive days. Five vehicle control animals were similarly treated, but with vehicle alone (Ethanol:water 7:3 v/v).
Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised.
After precipitating the DNA of the lymph node cells, radioactivity measurements were done.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks:

experimental

Value:

1

Test group / Remarks:

5% test substance

Results 2

Parameter:

SI

Remarks:

experimental

Value:

0.9

Test group / Remarks:

25% test substance

Results 3

Parameter:

SI

Remarks:

experimental

Value:

4

Test group / Remarks:

50% test substance

Results 4

Parameter:

SI

Remarks:

experimental

Value:

1

Test group / Remarks:

50% test substance

Results 5

Parameter:

SI

Value:

1

Test group / Remarks:

vehicle control, ethanol:water (7:3 v/v)

Applicant's summary and conclusion

Interpretation of results:

other: not sensitising

Conclusions:

The SI values calculated for the substance concentrations 5 and 25% were 1.0 and 0.9 respectively.
The SI of 4.0, calculated for the initially treated group at 50%, was not confirmed in the additionally group treated at 50% that showed a SI of 1.0. Based on the individual SI's, an overall SI of 2.5 was calculated.

There was no clear evidence that the test substance could elicit a SI >= 3.

Based on these results and according to the recommendations made in the test guidelines (OECD No.429, EC B.42 and EPA OPPTS 870.2600), A 094 should not be regarded as a skin sensitiser.

Based on these results and according to the:
- OECD Harmonized lntegrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances (OECD, 1998), A 094 does not have to be classified for sensitisation by skin contact.
- EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), A 094 does not have to be classified and has no obligatory labelling requirement for sensitisation by skin contact.

Executive summary:

Assessment for Contact Hypersensitivity to A 094 in the Mouse (Local Lymph Node Assay).

The study was carried out based on the guidelines described in: OECD, Section 4, Health Effects, No.429 (2002), Paris Cedex; EC, Council Directive 67/548/EEC, Annex IV C, B.42 (Draft) (2001 ); Environmental Protection Agency (EPA): Health Effects Test Guidelines OPPTS 870.2600. "Skin Sensitisation" 2003.

Test substance concentrations selected for the main study were based on the results of a preliminary study.

In the main study, three groups of five experimental animals were epidermally exposed to a 5%, 25% and 50% concentration respectivelyon three consecutive days. Five vehicle control animals were similarly treated, but with vehicle alone (Ethanol:water 7:3 v/v).

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised.

After precipitating the DNA of the lymph node cells, radioactivity measurements were done.

The majority of nodes were equal in size, except for the nodes of one animal treated at 50%. No other macroscopic abnormalities of the nodes were noted.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 25 and 50% were 111, 107 and 452 respectively. The mean DPM/animal value for the vehicle control group was 113.

In order to clarify the response at 50%, additional groups of animals were treated. A mean DPM/animal value for the experimental group treated with a test substance concentration of 50% was 372. The mean DPM/animal value for the vehicle control group was 381.

The SI values calculated for the substance concentrations 5 and 25% were 1.0 and 0.9 respectively.

The SI of 4.0, calculated for the initially treated group at 50%, was not confirmed in the additionally group treated at 50% that showed a SI of 1.0. Based on the individual Sl's, an overall SI of 2.5 was calculated.

There was no clear evidence that the test substance could elicit a SI >= 3.

Based on these results and according to the recommendations made in the test guidelines (OECD No.429, EC B.42 and EPA OPPTS 870.2600), A 094 should not be regarded as a skin sensitiser.

Based on these results and according to the:

- OECD Harmonized lntegrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances (OECD, 1998), A 094 does not have to be classified for sensitisation by skin contact.

- EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), A 094 does not have to be classified and has no obligatory labelling requirement for sensitisation by skin contact.