fermentation residues of molasses, yeast extract, sucrose and ammonia with bacillus subtilis

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 November 2009 - 14 December 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Principles of method if other than guideline:

not applicable

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:

Biomass residue (liquid) obtained after B2 production

IUPAC Name:

Biomass residue (liquid) obtained after B2 production

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:Charles River France, L¿Arbresle Cedex, France.
-Number of animals: 20 females (nulliparous and non-pregnant), five females per group.
-Age and bodyweight: Young adult animals (approx. 11 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.
- Housing: 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.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL 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 ± 3.0ºC (actual range: 17.1 ¿ 21.9ºC), a relative humidity of 40-70% (actual range: 44 - 73%) and 12 hours artificial fluorescent light and 12 hours darkness per day.

IN-LIFE DATES: From: 30 November 2009 To: 14 December 2009

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

0%, 25%, 50%, 100%

No. of animals per dose:

5

Details on study design:

Three experimental groups of five female CBA/J mice were treated with test substance concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Propylene glycol).
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.

Statistics:

Not applicable

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: %: SI ± SEM 0% (vehicle): 1.0 ± 0.4 25%: 1.2 ± 0.4 50%: 1.5±0.4 100%: 1.1±0.3

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: %: DPM ± SEM 0% (vehicle): 340 ± 85 25%: 392 ± 82 50%: 503±62 100%: 363±40

Any other information on results incl. tables

No irritation of the ears was observed in any of the animals examined. Brown staining by the test substance did not hamper the scoring for erythema. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted.

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant.

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

Reliability check:

The SI values calculated for the HCA concentrations 5, 10 and 25% were 1.4, 1.2 and 5.1 respectively. An EC3 value of 16.9% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 2 and 20%. Theresults of the 6 monthly HCA reliability checks of the recent years were13.1, 15.6, 14.1, 13.8, 13.9, 16.0 and 11.9%.

Based on the results, it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The SI values calculated for the substance concentrations 25, 50 and 100% were 1.2, 1.5 and
1.1 respectively.
Since there was no indication that the test substance elicit an SI = 3 when tested up to 100%,
Biomass residue (liquid) obtained after B2 production was considered to be a non skin
sensitizer
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 Biomass residue (liquid) obtained after B2 production would not be
regarded as skin sensitizer according to the recommendations made in the test guidelines and
does not have to be classified and has no obligatory labeling requirement for sensitization by
skin contact according to the Globally Harmonized System of Classification and Labeling of
Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on
classification, labeling and packaging of substances and mixtures.