[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin sensitisation, rabbit (OECD 429, LLNA): not sensitising

Read-across from structural analogue source substance glycerides, castor-oil mono-, hydrogenated, acetates (CAS 736150-63-3).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Apr - 24 Apr 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see Remark

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz, Wiesbaden, Germany

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA7CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, Horst, The Netherlands
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 19.4 ± 0.9 g
- Housing: animals were housed individually in Makrolon Type I cages with wire mesh top (EHRET GmbH, Emmedingen, Germany) and granulated soft wood bedding (Harlan Winkelmann GmbH, Borchen, Germany)
- Diet: pelleted standard diet (Harlan Winkelmann GmbH, Borchen, Germany), ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethyl sulphoxide

Concentration:

10, 20 and 50% (w/v)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Study design: 50% (Batch D) was tested in a non-GLP pre-experiment
- Compound solubility: insoluble in water, soluble in DMSO
- Lymph node proliferation response: DPM-Background (1 mL 5% trichloroacetic acid) per animal (2 lymph nodes) controls: 2707.1 (animal 1), 2357.9 (animal 2), 2199.6 (animal 3), 2336.3 (animal 4) and 3184.5 (animal 5); DPM-Background (1 mL 5% trichloroacetic acid) per animal (2 lymph nodes) test group: 6794.2 (animal 1), 5977.4 (animal 2), 6981.8 (animal 3), 7739.5 (animal 4), 6305.7 (animal 5)

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-methyl thymidine incorporation determined by β-scintillation and γ-counting, respectively
- Criteria used to consider a positive response: A test item was regarded as a sensitiser in the LLNA if the following criteria were fulfilled: The exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index. The data were compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression. The decision to select a stimulation index (SI) of 3 as an arbitrary indication of sensitising activity was made on the basis of investigations performed with a wide range of chemicals.

TREATMENT PREPARATION AND ADMINISTRATION: Each test group was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations. The application volume, 25 µL, was spread over the entire dorsal surface of each ear lobe once daily for 3 consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). Five days after the first topical application, all mice were administered with 250 µL of 80.6 µCi/mL ³HTdR (corresponds to 20.2 µCi ³HTdR per mouse) by intravenous injection via a tail vein. Five hours later, all mice were euthanized by intraperitoneal injection of Na-thiopental. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions in PBS of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with PBS the lymph node cells were resuspended in 5% trichloroacetic acid and incubated at approximately 4 °C for at least 18 h for precipitation of macromolecules. The precipitates were resuspended in 5% trichloroacetic acid and transferred to plastic scintillation vials.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables. An ANOVA (Kruskal Wallis) followed by a Dunnett-test or t-test (Mann-Whitney), when applicable, were conducted to assess whether the difference of DPM per animal was statistically significant between test item groups or the positive control (RCA) group and the negative control (vehicle) group. However, both biological and statistical significance were considered together.

Positive control results:

The positve control substance (25% hexyl cinnamic aldehyde in acetone:olive oil (4+1)) induced positive reactions in 2/5 animals (40%).The SI-values of the positive control were 2.3, 1.6, 2.6, 4.2 and 4.8, respectively.

Key result

Parameter:

SI

Value:

0.97

Test group / Remarks:

10% formulation

Key result

Parameter:

SI

Value:

1.53

Test group / Remarks:

20% formulation

Key result

Parameter:

SI

Value:

1.86

Test group / Remarks:

50% formulation

In the study, stimulation indices of at the maximum 0.97 in the 10% groups, 1.53 in the 25% groups and 1.86 in the 50% groups were observed. At a concentration of 50% of Batch A a statistically significant difference of DPM per animal was observed (p ? 0.05), however, the stimulation index (S.I.) was 1.71. EC3 values of the test groups could not be calculated, since none of the tested concentrations induced a S.I. greater than 3.

VEHICLE CONTROL DATA:

DPM values per lymph node obtained for the vehicle control were relatively high when compared to the historical control data. The results for the individual animals showed low variability and the results are nonetheless considered as reliable. Differences in vehicle control data when compared to other studies are related to the use of different batches of 3H-Thymidine. Experiments performed with the same batch of 3H-Thymidine as used in the present study have shown DPM values per lymph node, which were considerably higher than the results obtained with earlier and later batches. It is assumed that incorporation of 3H-Thymidine is more efficient with the present batch even though evidence for this hypothesis cannot be provided. Since all animals used for this study were labeled with the same 3H-Thymidine-solution this increase is supposed to be higher for all test groups and does neither affect the sensitivity of the assay nor the calculation of S.l. values.

Table 1. Results of the LLNA.

Test item concentration % (w/v)	Group	Calculation	DPM per lymph node	Result
		number of lymph nodes		S.I.
-	BG_I		37.1*
-	BG_II		17.89*
-	Control group	10	2027.9	1.0
25	Positive control Group	10	6312.4**	3.11
10	Test group Batch A	10	1528.3	0.75
25	Test group Batch A	10	2088.3	1.03
50	Test group Batch A	10	3468.7**	1.71
10	Test group Batch B	10	1964.4	0.97
25	Test group Batch B	10	3107.2	1.53
50	Test group Batch B	10	3768.4	1.86
10	Test group Batch C	10	1211.2	0.60
25	Test group Batch C	10	2295.3	1.13
50	Test group Batch C	10	2213.7	1.09
10	Test group Batch D	10	1225.7	0.60
25	Test group Batch D	10	1688.8	0.83
50	Test group Batch D	10	2868.6	1.41

* Measurement of DPM only.

** Mean DPM value for the group was according to the Dunnett test significanlty higher than the corresponding control value. The p value for the analysis was <0.05

BG = Background (1 mL 5% trichloroacetic acid) in duplicate

S.I. = Stimulation Index

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Conclusions:

CLP: not classified

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study performed according to internationally accepted testing guideline, well documented, without GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

Maximisation test [Magnusson B. and Kligman A. M. (1969) J. Invest. Dermatol., 52, 268-276.]

Deviations:

yes

Remarks:

no positive control results provided in study report

GLP compliance:

no

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was performed prior to the current amendment of Regulation (EC) No. 1907/2006 that specifies the Local lymphy node assay (LLNA) as the first-choice method for in vivo testing.

Species:

guinea pig

Strain:

other: albino

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: David Hall Limited, Burton-on-Trent, Staffordshire, U. K.
- Age: 8 – 12 weeks
- Weight at study initiation: 345 – 438 g
- Housing: housed in groups of up to four in solid floor polypropylene cages furnished with softwood sawdust
- Diet (e.g. ad libitum): guines pig diet, J.Waring Ltd (feeds), Shardlow, Derbyshire
- Water (e.g. ad libitum): The drinking water was supplemented daily with vitamin C tablets
- Acclimation period: not mentioned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3
- Humidity (%): not mentioned
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal and epicutaneous

Vehicle:

other: intradermal: liquid paraffin B.P.; topical: petroleum jelly B.P.

Concentration / amount:

Intradermal Induction: 5%
Topical Induction: 25%

Route:

epicutaneous, occlusive

Vehicle:

other: intradermal: liquid paraffin B.P.; topical: petroleum jelly B.P.

Concentration / amount:

Topical challenge 25%

No. of animals per dose:

test animals: 10 (main test); 6 (range finding)
control animals: 5

Details on study design:

RANGE FINDING TESTS: Intradermal Injection: An area of 4x6 cm at the shoulder region of 2 animals were clipped. Into these areas were injected simultaneousely four 0.1 ml intradermal injections of the test material with the following concentrations: guinea pig A: 1% and guinea pig B: 5% in paraffin. Animals were observed 24, 48, 72 h and 7 days following treatment. Topical Application: The test material at two concentrations, was topically applied under an occlusive patch to four sites on the shaved flanks of each of a group of two guinea pigs. These animals had previously been intradermally injected with FDA. Following a 24 h exposure period the patches were removed and the degrees of irritancy at the test sites were evaluated for erythema immediately after removal of the patches and then at 24 and 48 h. The concentrations of the test material to be used on two further animals were chosen, applied and then evaluated after similar time intervals.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1
- Exposure period: intradermal: 5% w/v in paraffin;
- Concentration in Freunds Complete Adjuvants (FCA): 5% in in 50% v/v FCA/paraffin
- Test group: dermal: test substance in paraffin
- Control group: paraffin
- Frequency of applications: induction on days 0 and 7
- Duration: removal of patches after 48 hours
- Concentrations: 5% w/v

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: on day 21
- Exposure period: dermal application for 24 hours
- Test groups: test substance in paraffin
- Control group: test substance in paraffin
- Site: flank-region
- Concentrations: 5% w/v
- Evaluation (hr after challenge): 24 and 48 hours after application

Positive control substance(s):

not specified

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

induction: 0%; topical challenge: 25%

No. with + reactions:

0

Total no. in group:

5

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

intradermal induction: 5%, topical induction 25%; topical challenge: 25%

No. with + reactions:

0

Total no. in group:

10

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Remarks on result:

other: positive control not specified in study report

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

induction: 0%; topical challenge: 25%

No. with + reactions:

0

Total no. in group:

5

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

intradermal induction: 5%, topical induction 25%; topical challenge: 25%

No. with + reactions:

0

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Remarks on result:

other: positive control not specified in study report

RESULTS OF PILOT STUDY: Guinea pig A (concentration 1%): 24, 48 and 72 h slight erythema, 7 days none. Guinea pig B, (concentration 5%): 24, 48, 72h and 7 days slight erythema.

RESULTS OF TEST

- Sensitization reaction: No animals of the test or control groups produced reactions at either the test or vehicle control sites, at the 24 or 48 h readings.

- Clinical signs: No clinical signs were reported.

- Grading system: according to the Magnusson and Kligman grading scale for the evaluation of challenge patch test reactions

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Conclusions:

CLP: not classified

Executive summary:

The sensitizing potential of the test substance (CAS 91744-38-6, Glycerides, C16-18 mono-, di- and tri-, hydrogenated, citrates, potassium salts) was investigated in a study according to OECD Guideline No. 406 "Skin Sensitization (maximization test)”.

The following numbers of guinea pigs were used in the study: 10 (main test); 6 (range finding), control animals: 5.

The substance concentrations for induction were 5 % (intradermal induction) and 25 % (topical induction). The concentration for topical challenge was 25 %. The vehicle for intradermal induction was liquid paraffin B.P. Petroleum jelly B.P. was used for topical induction and challenge.

Animal sites were graded according to the Magnusson and Kligman grading scale for the evaluation of challenge patch test reactions.

No animals of the test or control groups produced sensitization reactions at either the test or vehicle control sites, at the 24 or 48 h readings. No clinical signs were reported.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

key study

Justification for type of information:

refer to the analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Parameter:

SI

Value:

0.97

Test group / Remarks:

10% formulation

Remarks on result:

other: Source CAS 736150-63-3

Key result

Parameter:

SI

Value:

1.53

Test group / Remarks:

25% formulation

Remarks on result:

other: Source CAS 736150-63-3

Key result

Parameter:

SI

Value:

1.86

Test group / Remarks:

50% formulation

Remarks on result:

other: Source CAS 736150-63-3

In addition to the data of the key study with source substance CAS 736150 -63 -3 further data on skin sensitisation are available from a supporting study with source substance CAS 91744 -38 -6. The supporting information comprises data of a Guinea Pig Maximisation Test (GPMT), in which 10 animals were induced with 5% CAS 91744 -38 -6 intradermally and with 25% epicutaneously, followed by a topical challenge at 25%. None of the treated animals showed skin reactions at the first (24 h) or second (48 h) reading. The study with source substance CAS 91744 -38 -6 was considered to provide supporting information as no positive control was included.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Conclusions:

As detailed in the analogue justification, it is considered that the target and the source substances are unlikely to lead to differences in skin sensitisation potential. The available data on the suitable source substances CAS 736150-63-3 and CAS 91744-38-6 did not show any skin sensitising effects. Therefore, the target substance is not predicted to be a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Justification for read-across

There are no data on skin sensitisation available for glycerides, C16-18 (even numbered) mono- and di- and their citrates (EC 701-358-7). The assessment of skin sensitisation was therefore based on studies conducted with analogue substances as part of a read across approach, in order to fulfil the standard requirements set out in Annex VII, 8.3, in accordance with Annex XI, 1.5, of Regulation (EC) No. 1907/2006. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. For skin sensitisation, read-across was conducted with the structurally related analogue substances glycerides, castor-oil mono-, hydrogenated, acetates (CAS 736150-63-3)and glycerides, C16-C18 (even numbered) mono-, di- and tri-, hydrogenated, citrates, potassium salts (CAS 91744-38-6). A statement on the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

 

CAS No. 736150-63-3

The skin sensitising potential of Glycerides, castor-oil mono, hydrogenated, acetates was investigated in a Local Lymph Node Assay (LLNA) in mice performed according to OECD guideline 429 and in compliance with GLP (DuPont, 2007). In this study, 5 female CBA7CaOlaHsd mice per test group were treated with test substance at 10, 20 and 50% (w/v) in dimethyl sulfoxide or vehicle alone, respectively. The test substance formulations or the vehicle were applied topically to the dorsal surface of each ear lobe (25 µL/ear) for three consecutive days. Different batches (A-D) were used for treatment with the test substance at the respective concentrations. Five days after the first topical application, animals were sacrificed and the cell proliferation of pooled lymph nodes from individual animals was measured by incorporation of ³H-methyl thymidine and expressed as the amount of radioactive disintegration per minute (DPM). For batch A, the mean DPM/lymph node for each test group was 1528.3, 2088.3 and 3468.7 at concentrations of 10, 20 and 50% of the test substance, respectively. At 50% concentration of Batch A, a statistically significant difference (p ≤ 0.05) in DPM per lymph node was observed compared to control (DPM/node = 2027.9). Based on these results, stimulation indices of 0.75, 1.03 and 1.71 were calculated for the treatment concentrations of 10, 20 and 50%, respectively. The animals treated with the mid and the highest concentration (25% and 50%) of Batch A showed reddening of the ear skin from the second day of treatment. In summary, stimulation indices at of maximal 0.97 in the 10% groups, 1.53 in the 20% groups and 1.86 in the 50% groups were observed after treatment with the batches A-D of the test substance. Based on this data, no EC3 values of the test groups could be calculated, since none of the tested concentrations induced a stimulation index greater than 3. The positive control substance (25% hexyl cinnamic aldehyde in acetone:olive oil (4+1)) induced positive reactions in 2/5 animals (40%) and thus confirmed the sensitivity and reliability of the experimental technique. Under the above mentioned conditions, the test substance was not found to be a sensitiser in the LLNA.

CAS 91744-38-6

The sensitising potential of glycerides, C16-18 mono-, di- and tri-, hydrogenated, citrates, potassium salts was studied in guinea pigs according to the maximisation method (OECD guideline 406) and in compliance with GLP (Sasol, 1981). The following numbers of guinea pigs were used in the study: 10 (main test); 6 (range finding), control animals: 5. A concentration of 5% of the test substance was used for intradermal induction and 25% was used for topical induction. The concentration used for the topical challenge phase was 25%. The vehicle used for intradermal induction was liquid paraffin B.P. Petroleum jelly B.P. was the vehicle used for the topical induction and challenge phases. Test sites were graded according to the Magnusson and Kligman grading scale for the evaluation of challenge patch test reactions. No animals of the test or control groups produced sensitization reactions at either the test or vehicle control sites, at the 24 or 48 h readings. No clinical signs were reported. Therefore, the test substance had no sensitising effect in guinea pigs under the experimental conditions chosen. As no positive control group was included in the study, the results can be used as an indication only and not for classification purposes.

 

Overall conclusion for skin sensitisation

The reliable data available for the read-across analogue substances glycerides, C16-18 and C18-hydroxy mono- and di- (CAS No. 91845-19-1) and glycerides, C16-C18 (even numbered) mono-, di- and tri-, hydrogenated, citrates, potassium salts / glycerides, C16-18 (even numbered) mono-, di- and tri-, hydrogenated, citrates, potassium salts (CAS No. 91744-38-6) did not show any skin sensitising effects. Therefore, glycerides, C16-18 (even numbered) mono- and di- and their citrates (EC 701-358-7) is not expected to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on read-across from structurally similar substances, the available data on skin sensitisation do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.