Esterification product of castor oil and tetrahydromethyl-1,3-isobenzofuranedione

Endpoint summary

Description of key information

REACH_sensitising | mice (female) | OECD 429 | #key study#

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May-June 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

April 2002

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

April 2004

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Hygroscopic: yes
- Specific Gravity: 1.10 g/cc

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

ANIMALS
- Mouse, CBA strain, inbred, SPF-Quality; recognized by the international guidelines as the recommended test system (e.g, OECD, EC, EPA).
- Source: Charles River France, L'Arbresle Cedex, France
- 20 females (nulliparous and non-pregnant), five females per group.
- Young adult animals (approx. 9 weeks old) were selected.
- Body weight variation was within +/- 20% of the sex mean.
- Identification: Tail mark with marker pen.
A health inspection was performed prior to treatment, to ensure that the animals are in a good state of health, Special attention was paid to the ears, which were intact and free from any abnormality.

HUSBANDRY
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: 20.2-24,0°C), a relative humidity of 30-70% (actual range: 43 - 95%) and 12 hours artificial fluorescent light and 12 hours darkness per day.
Accommodation: Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd). Surrey, United Kingdom) was supplied as cage-enrichment.
The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.
Acclimatization period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. Accommodation was as described above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm).
Diet: Free access to pelleted rodent diet (SM R/M-Z Irom SSNIFF® Spezialdia1:en GmbH, Soest, Germany).
Water: Free access to tap water.
Results of analysis lor each batch of diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

Vehicle:

dimethylformamide

Concentration:

0, 25, 50, 100 %

No. of animals per dose:

5

Details on study design:

In the main study, three groups of five experimental animals were treated with test substance concentrations of 25%, 50% or 100% on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Dimethyl formamide).
Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) Iymph nodes were excised.
After precipitating the DNA of the Iymph node cells, radioaetivity measurements were performed. The activity was expressed as the number of Disinregrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated lor each group.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity. An EC3 value of 13.8 % was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 2 and 20%.

Key result

Parameter:

SI

Value:

7.2

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

11.8

Test group / Remarks:

50%

Key result

Parameter:

SI

Value:

13.3

Test group / Remarks:

100%

Parameter:

other: Mean DPM/animal

Value:

4 029

Test group / Remarks:

25%

Parameter:

other: Mean DPM/animal

Value:

6 576

Test group / Remarks:

50%

Parameter:

other: Mean DPM/animal

Value:

7 413

Test group / Remarks:

100%

Parameter:

other: Mean DPM/animal

Value:

557

Test group / Remarks:

vehicle control

Skin reactions / Irritation:

No irritation of the ears was seen in the control group and the 25% test substance treated group. The slight irritation of the ears as shown by the animals of the 50 and 100% treated groups was considered not to have a toxicologically significant effect on the activity of the nodes.

Macroscopy of the auricular lymph nodes and surrounding area:

All nodes of the experimental animals treated at 25% and control group were considered normal in size. Enlarged nodes were found in the groups treated at 50 and 100%. No macroscopic abnormalities of the surrounding area were noted.

Body weights:

The slight body weight loss, noted in some experimental animals, was considered not toxicologically significant.

Toxicity and mortality:

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The SI values calculated for the substance concentrations 25, 50 and 100% were 7.2, 11.8 and 13.3 respectively.
These results indicate that the test substance could elicit a SI >= 3. No reliable EC3 value could be calculated. It would have been possible to strengthen the outcome of the study by adding lower concentrations. Since the SI values clearly exceeded 3 and since extension of the study would not alter the classification, this was considered not appropriate for ethical reasons.
The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.
Based on these results:
- according to the recommendations made in the test guidelines, the Substance would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007), the Substance should be classified as skin sensitizer (Category 1).

Executive summary:

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 treated with test substance concentrations of 25%, 50% or 100% on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Dimethyl formamide).

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 performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No irritation of the ears was seen in the control group and the 25% test substance treated group. The slight irritation of the ears as shown by the animals of the 50 and 100% treated groups was considered not to have a toxicologically significant effect on the activity of the

nodes.

All nodes of the experimental animals treated at 25% and control group were considered normal in size. Enlarged nodes were found in the groups treated at 50 and 100%.

The slight body weight loss, noted in some experimental animals, was considered not toxicologically significant.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 4029, 6576 and 7413 respectively. The mean DPM/animal value for the vehicle control group was 557.

The SI values calculated for the substance concentrations 25, 50 and 100% were 7.2, 11.8 and 13.3 respectively. These results indicate that the test substance could elicit a SI >= 3. No reliable EC3 value could be calculated. It would have been possible to strengthen the outcome of the study by adding lower concentrations. Since the SI values clearly exceeded 3 and since extension of the study would not alter the classification, this was considered not appropriate for ethical reasons.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The SI values calculated for the substance concentrations 25, 50 and 100% were 7.2, 11.8 and 13.3 respectively. No reliable EC3 value could be calculated. It would have been possible to strengthen the outcome of the study by adding lower concentrations. Since the SI values clearly exceeded 3 and since extension of the study would not alter the classification, this was considered not appropriate for ethical reasons. It is estimated by linear correlation that the EC3 value would be clearly >2 %. The substance needs to be classified as Skin Sens 1B (H317) according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Classification is affected by the constituent Tetrahydromethylphtalic anhydride (EC: 234 -290 -7, CAS: 11070 -44 -3) which is a respiratory sensitizer and listed under Annex VI of the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. Thus the substance needs to be classified as Resp. Sens 1 (H334) according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.