Amines, rosin

Endpoint summary

Description of key information

A local lymph node assay was performed in mice, in order to assess the skin sensitisation potential of the test substance, Rosin Amine 90, in a procedure compatible with OECD Guideline 429 and Method B.42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008.

 

CBA/Ca mice were used in this study. Mice were randomly allocated to cages and randomly provided with unique study numbers. All animals were examined for observable skin lesions prior to treatment. The test substance was freshly prepared as a solution in butanone (vehicle) and applied topically to the dorsal surface of the ear within 2 hours of formulation. α‑Hexylcinnamaldehyde was used as a positive control in the assay, since it has been shown to produce the required dose response expected form a moderate sensitiser in historical positive control studies. The positive control was freshly prepared as a 15% v/v dilution in butanone.

A preliminary screening test was conducted on four mice, who were treated daily with 25 µL of test substance at concentrations 1%, 10%, 25% and 50% v/v in butanone, applied to the dorsal surface of each ear for up to three consecutive days (Days 1 -3). One mouse was used per test substance concentration. The mice were observed twice daily on Days 1 -3 and once daily on Days 4 -6. Local signs of irritation were scored daily, and any signs of clinical toxicity were noted. Body weight was recorded on Day 1 (prior to dosing) and on Day 6 of surviving mice. One mouse from the 25% Rosin Amine 90 v/v in butanone treatment group was humanely killed on Day 4 due to excessive weight loss (~18%), since it was considered to approach the moderate severity limit set forth in the UK Home Office Project License. The body weight of this animal was recorded immediately prior to termination. Ear thickness was also measured pre- and post-dose on Day 1, post-dose on Days 2 and 3 and on Days 4, 5 and 6. Mean ear thickness changes were calculated between Days 1 -3 and Days 4 -6 and a mean increase greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

 

The preliminary screening showed that Rosin Amine 90 did not produce systemic toxicity or excessive local skin irritation at 1% v/v in butanone, which was therefore selected as the highest suitable concentration. Groups of five mice per dose were treated with the test substance, at concentrations of 0.1%, 0.5% and 1% v/v in butanone. 25 µL of test substance was applied to the dorsal surface of each ear for three consecutive days (Days 1 -3) using an automatic micropipette. A concurrent group of five mice receive the vehicle (undiluted butanone) in the same manner. Five days following the first topical application of the test substance, all mice were injected via the tail vein with 250 µL of PBS containing 3H-methyl thymidine, giving a total of 20 µCi to each mouse. Mice were observed twice daily from Day 1 -3 and daily from Day 4 -6 for signs of toxicity or ill health. Ear thickness and body weight were measured in the same manner as during the preliminary screening test. Five hours following 3HTdR administration, all mice were killed by carbon dioxide asphyxiation. Lymphocyte proliferation was measured in the lymph nodes draining from the site of test substance application during the induction phase of skin sensitisation. The Stimulation Index (SI) was determined as a ratio of the mean proliferation in each treated group to that in the concurrent vehicle control (VC) group. An SI ≥3 warrant the classification of the test substance as a potential skin sensitizer.

There were no signs of systemic toxicity noted in the treatment or control groups and body weight change of test animals between Day 1 -6 was comparable to that seen in the corresponding control group animals over the same period. Positive results were seen in the estimation of proliferative response of lymph node cells for 0.5% (SI = 21.31) and 1% (SI = 45.13) v/v in butanone, whilst 0.1% v/v Rosin Amine 90 in butanone gave a negative result (SI = 1.34).

The positive control item also gave a positive result, as expected (SI = 6.09). Rosin amine 90 at 0.13% v/v in butanone was calculated to be the concentration expected to cause a 3 -fold increase in 3HTdR incorporation (EC3 value).

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:

30th November 2017 - 6th February 2018

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 20170314028
- Expiration date of the lot/batch: 01 March 2019
- Purity: 100% (UVCB)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: It was assumed that the test substance formulation in the vehicle was stable for the duration of exposure.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was freshly prepared, as required, as a solution in butanone.
- It was formulated within 2 hours of being applied to the test system.
- No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: 15-23g
- Housing: suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study
- Water (e.g. ad libitum): free access to mains tap water
- Acclimation period: at least 5 days
- Indication of any skin lesions: prior to the start of treatment, animals were examined to ensure that they had no observable skin lesions. Any animals found with adverse effects that were considered to approach the moderate severity limit set forth in the UK Home Office Project Licence, were humanely killed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Air changes (per hr): at least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12hrs dark/12 hrs light
- Enrichment: animals were provided with environmental enrichment items that were not considered to contain a signficant level of contaminants that would affect the purpose of integrity of the study.

Vehicle:

other: Butanone

Remarks:

This vehicle was chosen as it produced the highest concentration that was suitable for dosing.

Concentration:

Preliminary screening test: 1%, 10%, 25%, 50% v/v Rosin Amine 90 in butanone
Main test: 0.1%, 0.5%, 1% v/v Rosin Amine 90 in butanone

No. of animals per dose:

Preliminary screening test: 4 animals, one animal per test substance concentration
Main test: 5 animals per dose

Details on study design:

PRE-SCREEN TESTS:
- Mice were treated by daily application of 25 µl if the test substance formulation to the dorsal surface of each ear for up to three consecutive days (Days 1, 2, 3)
- Mice were observed twice daily on Days 1-3 and once daily on Days 4-6 for surviving mice.
- Irritation: local irritation was socred daily according the the scale
- Systemic toxicity: clinical signs of toxicity, if present, were recorded
- Body weight: recorded on Day 1 (prior to dosing) and on Day 6 (for surviving mice)
- Ear thickness measurements: mean thickness was measured using a Mitutoyo 547-300S gauge, pre- and post-dose on Day 1, post-dose on Days 2-6. Any changes in ear thickness were noted. Mean ear thickness changes were calculated between Days 1-3 and Days 4-6 and an increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

MAIN STUDY
- The preliminary screening test showed that the test item did not produce systemic toxicity or excessive local skin irritation at 1% v/v in butanone, which was the highest suitable concentration.
- The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).
- The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
- A concurrent group of five mice received the vehicle butanone (undiluted) in the same manner.
- Five days following the first topical application of the test item or vehicle on Day 6 all mice were injected via the tail vein with 0.25 mL (250 µL) of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.

ANIMAL ASSIGNMENT AND TREATMENT
- Identification: on receipt, the animals were randomly allocated to cages and given a number unique within the study by indelible ink markings on their tails and a number written on the cage cards.
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in3HTdR incorporation will be classified as a "non-sensitizer".

Positive control substance(s):

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

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships.
Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non parametric Kruskal Wallis Rank Sum and Mann Whitney U test procedures were used.

Probability values (p) are presented as follows:
P<0.001 = ***
P<0.01 = **
P<0.05 = *
P>0.05 = (not significant)

Positive control results:

Stimulation index = 6.09 (positive result)

Key result

Parameter:

SI

Value:

ca. 1.34

Test group / Remarks:

0/1% v/v in butanone

Remarks on result:

other: Negative result

Key result

Parameter:

SI

Value:

ca. 21.31

Test group / Remarks:

0.5% v/v in butanone

Remarks on result:

other: Positive result

Key result

Parameter:

SI

Value:

ca. 45.13

Test group / Remarks:

1% v/v in butanone

Remarks on result:

other: Positive result

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION : The Stimulation Index expressed as the mean radioactive incorporation for each treatment group was divided by the mean radioactive incorporation of the vehicle control group.

EC3 CALCULATION : The concentration of test item, Rosin amine 90, expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 0.13%.

CLINICAL OBSERVATIONS: There were no signs of systemic toxicity noted in the Rosin amine 90 exposed or control animals during the test

BODY WEIGHTS: Body weight change of the test anumals between Day 1-6 was comparable to that observed in the corresponding control group animals over the same period.

Preliminary screening test

- One animal treated in the 25% Rosin Amine 90 v/v in butanone group was humanely killed on Day 4 due to excessive weight loss (~18%), since it was considered to approach the moderate severity limit set forth in the UK Home Office Project License.

- No signs of systemic toxicity or visual local skin irritation were noted in the animals treated with the test item at a concentration of 10% or 50% v/v in butanone but irritation indicated by a greater than 25% increase in mean ear thickness was noted in both animals.

- No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness was noted in the animal treated with the test item at a concentration of 1% v/v in butanone.

- Based on this information the dose levels selected for the main test were1%, 0.5% and 0.1% v/v in butanone.

Calculation of the EC3 value

EC₃= c + [[(3-d)/(b-d)] x (a-c)]

a	=	0.5
b	=	21.31
c	=	0.1
d	=	1.34
EC3=0.1+ [[(3-1.34)/(21.31-1.34)] x (0.5-0.1)] =0.13

The concentration of test item, Rosin amine 90, expected to cause a 3 fold increase in³HTdR incorporation (EC₃value) was calculated to be0.13%.

a=  lowest concentration giving stimulation index >3

b = actual stimulation index caused by ‘a’

c =  highest concentration failing to produce a stimulation index of 3

d = actual stimulation index caused by ‘c’

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

Based on the results of the study, the test substance, Rosin Amine 90, was considered to be a sensitiser under the conditions of the test and classified as a contact sensitiser (Category 1A) according to the Globally Harmonised System of Classification and Labelling of Chemicals (GHS) and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures (CLP). Therefore, the signal word 'Warning' and the Hazard Statement 'H317: May cause an allergic skin reaction' is required.

Executive summary:

The study was performed in order to assess the skin sensitisation potential of the test substance, Rosin amine 90, via a mouse local lymph node assay in a procedure compatible with OECD Guideline 429 and Method B.42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008.

 

CBA/Ca mice were employed in this study, since they have been previously shown to produce satisfactory responses using known sensitizers and non‑sensitizers during the in‑house validation. They were randomly allocated to cages and randomly provided with unique study numbers. All animals were examined for observable skin lesions prior to treatment. The test substance was freshly prepared as a solution in butanone (vehicle) and applied topically to the dorsal surface of the ear within 2 hours of formulation. α‑Hexylcinnamaldehyde was used as a positive control, which was freshly prepared as a 15% v/v dilution in butanone, since it has been shown to produce the required dose response expected form a moderate sensitiser in historical positive control studies.

 

A preliminary screening test was conducted on four mice, who were treated by daily application of 25 µL of test substance at concentrations 1%, 10%, 25% and 50% v/v in butanone to the dorsal surface of each ear for up to three consecutive days (Days 1 -3). One mouse was used per test substance concentration. The mice were observed twice daily on Days 1 -3 and once daily on Days 4 -6. Local signs of irritation were scored daily and any signs of clinical toxicity were noted. Body weight was recorded on Day 1 (prior to dosing) and on Day 6 of surviving mice. One mouse from the 25% Rosin Amine 90 v/v in butanone treatment group was humanely killed on Day 4 due to excessive weight loss (~18%), since it was considered to approach the moderate severity limit set forth in the UK Home Office Project License. The body weight of this animal was recorded immediately prior to termination. Ear thickness was also measured pre- and post-dose on Day 1, post-dose on Days 2 and 3 and on Days 4, 5 and 6. Mean ear thickness changes were calculated between Days 1 -3 and Days 4 -6 and a mean increase greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

 

The preliminary screening showed that Rosin Amine 90 did not produce systemic toxicity or excessive local skin irritation at 1% v/v in butanone, which was therefore selected as the highest suitable concentration. Groups of five mice per dose were treated with the test substance, at concentrations of 0.1%, 0.5% and 1% v/v in butanone. 25 µL of test substance was applied to the dorsal surface of each ear for three consecutive days (Days 1 -3) using an automatic micropipette. A concurrent group of five mice receive the vehicle (undiluted butanone) in the same manner. Five days following the first topical application of the test substance, all mice were injected via the tail vein with 250 µL of PBS containing 3H-methyl thymidine, giving a total of 20 µCi to each mouse. Mice were observed twice daily from Day 1 -3 and daily from Day 4 -6 for signs of toxicity or ill health. Ear thickness and body weight were measured in the same manner as during the preliminary screening test. Five hours following 3HTdR administration, all mice were killed by carbon dioxide asphyxiation . Lymphocyte proliferation was measured in the lymph nodes draining from the site of test substance application during the induction phase of skin sensitisation. The Stimulation Index (SI) was determined as a ratio of the mean proliferation in each treated group to that in the concurrent vehicle control (VC) group. An SI ≥3 warrant the classification of the test substance as a potential skin sensitizer.

 

There were no signs of systemic toxicity noted in the treatment or control groups and body weight change of test animals between Day 1 -6 was comparable to that seen in the corresponding control group animals over the same period. Positive results were seen in the estimation of proliferative response of lymph node cells for 0.5% (SI = 21.31) and 1% (SI = 45.13) v/v in butanone, whilst 0.1% v/v Rosin Amine 90 in butanone gave a negative result (SI = 1.34). The positive control item also gave a positive result, as expected (SI = 6.09). Rosin amine 90 at 0.13% v/v in butanone was calculated to be the concentration expected to cause a 3 -fold increase in 3HTdR incorporation (EC3 value).

Based on the results of the assay and according the the data evaluation criteria, the test substance, Rosin amine 90, was considered to be a sensitiser. According to the Globally Harmonised System of Classification and Labelling of Chemicals (GHS) and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances ad Mixtures (EU CLP), the test substance was classified as a contact sensitiser (Category 1A) and therefore necessitates the signal word 'warning' and the hazard statement 'H317: May cause an allergic skin reaction'.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Justification for classification or non-classification

Skin sensitisation

Rosin amine 90, was considered to be a sensitiser. According to the Globally Harmonised System of Classification and Labelling of Chemicals (GHS) and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances ad Mixtures (EU CLP), the test substance was classified as a contact sensitiser (Category 1A) and therefore necessitates the signal word 'warning' and the hazard statement 'H317: May cause an allergic skin reaction'.