2-Butyne-1,4-diol, polymer with methyloxirane

Endpoint summary

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:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2010)

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)

Species:

mouse

Strain:

other: CBA/CaCrl

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Animal strain: mouse; CBA/CaCrl (Pre-test: CBA/CaOlaHsd)
- Source: Charles River, UK (Pre-test: Harlan Winkelmann GmbH, Germany)
- Age at study initiation: 8 - 11 weeks
- Mean weight at study initiation: ca. 16.2 - 20.5 g
- Housing: 5/cage; Makrolon cages, type II/III
- Diet: Pelleted standard diet, Harlan Laboratories B.V., Horst, Netherlands; ad libitum
- Water: Tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 35- 65
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

50%, 25% and 10%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which could be technically used, was 100%. The dilutions were formulated in acetone/olive oil/4:1 (v/v) (AOO).
To determine the highest non-irritant test concentration that does not induce signs of systemic toxicity at the same time, a pre-test was performed in two animals. Two mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 50% (w/w) and 100% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible reddening of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if reddening of the ear skin of a score value V3 was observed at any observation time and/or if an increase in ear thickness of V25% was recorded on day 3 or day 6. The measured ear weight was also considered in this evaluation.
At the tested concentrations the animals did not show any signs of systemic toxicity. On day 4 and day 5, the animal treated with the undiluted test item showed an erythema of the ear skin (Score 1). Other signs of irritation or signs of systemic toxicity were not observed.
Additionally, at both tested concentrations, an increase in ear weight was observed that exceeded the threshold value of 25% for excessive local skin irritation mentioned in OECD guideline 429. Measurement of ear thickness did not confirm the ear weight, but was increased at 100% (+ 18.9%). Thus, the test item in the main study was assayed at 10, 25, and 50% (w/w).

MAIN STUDY
TREATMENT SCHEME
Vehicle control group 1: AOO
Test group 2: 10% test item in AOO
Test group 3: 25% test item in AOO
Test group 4: 50% test item in AOO

EXPERIMENTAL PROCEDURE
- Route of application: Epicutaneously to the dorsum of both ears
- Application volume: 25 μL per ear
- Site of application: Dorsal surface of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- Administration of 3H-Methyl Thymidine: Five days after the first topical application 250 µL of phosphate-buffered saline (PBS) containing 20.2 µCi of 3HTdR (equivalent to approximately 80.9 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
- Determination of Incorporated 3HTdR: Approximately five hours after treatment with 3HTdR all mice were sacrificed. The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared. The level of 3HTdR incorporation was measured on a beta-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). 3HTdR incorporation was expressed as the number of radioactive disintegrations per minute (DPM).
- Determination of Lymph Node Weight and Cell Count: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. Furthermore, the lymph node cell count was determined for each animal using a cell counter.
- Determination of ear weight: After the lymph nodes were excised, both ears of mice were punched at the apical area using a biopsy punch. For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.
- Evaluation of results: The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.).

CLINICAL EXAMINATIONS
- Body weight determination: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: after the first application and prior to treatment with 3HTdR.
- Signs and symptoms: Obvious signs of systemic toxicity and/or local inflammation at the application sites were checked individually.
- Mortality: A check for moribund and dead animals was made at least once each workday.

CALCULATION OF RESULTS
- Results for each treatment group are expressed as the mean SI. The SI is derived by dividing the mean 3HTdR labelling index/mouse within each test substance group by the mean 3HTdR labelling index for the negative control (NC) group. The average SI for the NCs is then one.
- A result is regarded as positive when SI ≥ 1.55.

Positive control substance(s):

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

Statistics:

Calculation of mean and standard deviation was performed.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Test group 2 (10%): mean: 3.58 (individual values: 2.6, 5.1, 4.6, 2.0, and 3.6) Test group 3: (25%): mean: 9.94 (individual values: 7.5, 11.9, 10.4, 11.5, and 8.5) Test group 4: (50%): mean: 9.83 (individual values: 6.9, 10.5, 11.0, 10.4, and 10.4) SI-value of historical positive control: 8.08 at 25% alpha hexylcinnamaldehyde

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Test group 1 (vehicle control; NC): mean: 1127.8 (individual values: 1317, 1594, 1038, 741, and 1059) Test group 2 (10%): mean: 4041.8 (individual values: 2988, 5750, 5192, 2301, and 4088) Test group 3 (25%): mean: 11209.2 (individual values: 8462, 13396, 11719, 13026, and 9553) Test group 4 (50%): mean: 11081.0 (individual values: 7792, 11892, 12424, 11711, and 11696)

Table 1 Lymph Node Cell Count

Test item concentration % (w/w)	Group Calculation
	Mean lymph node cell count (x10E06 per group)	Standard deviation	Index
Vehicle (AOO)	9.78	1.93	1.0
10 % test substance	19.28*	5.81	1.97
25 % test substance	31.83*	4.92	3.25
50 % test substance	33.42*	5.69	3.42

Index = values of the test item groups related to the mean value of the control group

* statistically significant increase in comparison to vehicle control group (p ≤ 0.05)

 

Table 2 Lymph Node Weight

Test item concentration % (w/w)	Group Calculation
	Mean Lymph Node Weight (mg per group)	Standard deviation	Index
Vehicle (AOO)	6.15	0.79	1.0
10 % test substance	9.29*	1.17	1.51
25 % test substance	13.11*	1.58	2.13
50 % test substance	13.90*	2.01	2.26

Index = values of the test item groups related to the mean value of the control group

* statistically significant increase in comparison to vehicle control group (p ≤ 0.05)

 

Table 3 Ear Weights

Test item concentration % (w/w)	Group Calculation
	Mean ear weight (mg per group)	Standard deviation	Index
Vehicle (AOO)	24.89	3.40	1.0
10 % test substance	27.46	2.38	1.10
25 % test substance	34.29*	4.08	1.38
50 % test substance	47.76*	9.71	1.92

Index = values of the test item groups related to the mean value of the control group

* statistically significant increase in comparison to vehicle control group (p ≤ 0.05)

 

Further observations:

No deaths occurred during the study period. No systemic findings were observed during the study period. On day 4 and day 5, the animals treated with 25 and 50% of the test item showed an erythema of the ear skin (score 1). The animals treated with 50% test item additionally showed an erythema of the ear skin (score 1) on day 6. Animals treated with 10% test item did not show any sign of local skin irritation.

The body weight of the animals was within the range commonly recorded for animals of this strain and age.  

Interpretation of results:

sensitising

Remarks:

Migrated information

Executive summary:

In this study the test item Golpanol BEO was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item solution at different concentrations was prepared in the vehicle acetone:olive oil (4+1 v/v). The local lymph node assay is recommended by international test guidelines (e.g. OECD) as an animal test for predicting skin sensitization in humans and provides a rational basis for risk assessment. The basic principle underlying the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is termed the Stimulation Index (S.I.). Radioactive labelling is used to measure cell proliferation. For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, and 50% (w/w). The highest concentration tested was the highest concentration that could be technically used and applied whilst avoiding systemic toxicity and excessive local skin irritation (as determined by a pre-experiment).

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On day 4 and day 5, the animals treated with the test item concentrations of 25 and 50% showed an erythema of the ear skin (Score 1). The animals treated with 50% test item concentration additionally showed an erythema of the ear skin (Score 1) on day 6. Animals treated with 10% test item concentration did not show any signs of local skin irritation. A statistically significant and biologically relevant increase in ear weights was observed in the mid and the high dose group in comparison to the vehicle control group (p<0.05). Additionally, the cutoff-value for a positive response regarding the ear weight index of 1.1 reported for BALB/c mice (see Ref. 9) was exceeded in all dose groups. Furthermore, according to OECD guideline 429, an increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation. This threshold was exceeded in in the mid and high dose group (37.8 and 91.9%, respectively), thus indicating the irritant properties of the test item in the mid and high dose group, but not in the low dose group.

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices (S.I.) of 3.58, 9.94, and 9.83 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in acetone:olive oil (4+1 v/v), respectively. The EC3 value could not be calculated, since all S.I.s are above the threshold of 3. An outlier was identified in the high dose group (DPM value determined for animal number 16). However, as exclusion of the outlier did not change the overall test result, the value in question was not excluded from calculation. A statistically significant and biological relevant increase in DPM value and also in lymph node weight and cell count was observed in all dose groups in comparison to the vehicle control group. Furthermore, the cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice (see Ref. 8) was exceeded in all dose groups (indices of 1.97, 3.25, and 3.42, respectively) (BASF, 2012).

Based on the above mentioned findings regarding ear skin irritation, an influence of irritation on lymphocyte proliferation cannot be excluded, but only in the mid and high dose group. Nevertheless, on the basis of the present data, the test item has to be classified as

a sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

1.    Justification for grouping of substances and read-across

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

 

	Target Substance	Source Substance No 1	Source Substance No 2
Name	2-Butyne-1,4-diol, comp. with methyloxirane	2-Butyne-1,4-diol, comp. with oxirane (Polyethylene glycol ether with 2-butyne-1,4-diol)	2-Propyn-1-ol, compd. with methyloxirane
CAS	61596-96-1	32167-31-0	38172-91-7
Skin sensitization	--	OECD 429, sensitizing (BASF, 2012) (RL1)	OECD 429, not sensitizing (BASF, 2012), a.i. 54.7% applied up to 100% testitem

 

2.    Analogue approach justification

 

The above mentioned substances are considered to be similar on the basis of structural similarity resulting in similar properties and/or activities. The available endpoint information is used to predict the same endpoint for2-Butyne-1,4-diol, polymer with methyloxirane (CAS 61596-96-1).

Since no studies are available for2-Butyne-1,4-diol, polymer with methyloxirane (CAS 61596-96-1)investigating the skin sensitization,a read-across from the structurally related analogue substancePolyethylene glycol ether with 2-butyne-1,4-diol (CAS 32167-31-0)was usedin accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5.

 

3.    Result:

 

OECD-Guideline studies

Source substance No 1(CAS 32167-31-0)

 

LLNA:

 

In this study the test item was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice according to OECD 429 under GLP conditions(BASF, 2012). Test item solution at different concentrations was prepared in the vehicle acetone:olive oil (4+1 v/v) using concentrations of 10, 25, and 50% (w/w). The highest concentration tested was the highest concentration that could be technically used and applied whilst avoiding systemic toxicity and excessive local skin irritation (as determined by a pre-experiment). The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. Animals treated with 10% test item concentration did not show any signs of local skin irritation, whereas higher concentrations showed local irritation, whereas an increase in ear weight exceeding the threshold value of 25% was measured in the mid and high dose group (37.8 and 91.9%, respectively), thus indicating the irritant properties of the test item.

 

A test item is regarded as a sensitizer in the LLNA, if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.).

In this study Stimulation Indices (S.I.) of 3.58, 9.94, and 9.83 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in acetone:olive oil (4+1 v/v), respectively. The EC3 value could not be calculated, since all S.I.s are above the threshold of 3. A statistical increase above the cut off value can be found for the lowest (not irritating) concentration (10%). Furthermore, the cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was exceeded in all dose groups (indices of 1.97, 3.25, and 3.42, respectively).

 

Based on the above mentioned findings regarding ear skin irritation, an influence of irritation on lymphocyte proliferation cannot be excluded, but only in the mid and high dose group. On the result of the lowest does group, the test item has to be classified as a sensitizer.

 

 

Source Substance No 2 (CAS 38172-91-7)

One study is investigating the skin sensitizing potential is available for the source substance of 2-Propyn-1-ol, compd. with methyloxirane (CAS 38172-91-7). In order to study a possible skin sensitization potential of 2-Propyn-1-ol, compd. with methyloxirane (a.i. 54,7%), 3 groups each of 5 female mice were treated once daily with the test item at concentrations of 25, 50% and 100% (test item, a.i. 54.7%) in dimethylformamide by topical application to the dorsum of each ear for three consecutive days according to OECD 429 in compliance with GLP. The Stimulation Indices (S.I.) of 0.60, 1.14, and 0.71 were determined with the test item at concentrations of 25, 50% (w/w) and 100% in dimethylformamide, respectively. For higher active ingredient concentration a S.I. above 1.5 is not expected, as there is no increase in stimulation indices observed. Hence the source substance is not a skin sensitizer.

 

 

4.    Key study assignment:

As there are two studies with contrary results available. The result for the closer homologue (source substance 1) is used as a worst case assumption and classification is derived from this, nevertheless both studies are marked as WoE as it stays unclear if the positive result may be an artificial result as irritation was also observed, and the second source substance contains the same structure elements. 

 

5.    Conclusion

The cutoff-value for a positive response was exceeded for the lowest (not irritating (25%)) concentration for a close homologue to the target substance. The available data for the source substance 2-Butyne-1,4-diol, comp. with oxirane (CAS 32167-31-0) indicate a skin sensitizing potential, which is also proposed for the target substance 2-Butyne-1,4-diol, polymerwith methyloxirane (CAS 61596-96-1)as a worst case assumption. Nevertheless, there is also a negative LLNA study available for the source substance No 2 which contains similar structure element like the target and other source substance. This may question the results of the LLNA with source substance No 1 especially as there are some effects maybe also related an irritating effect and irritation is a confounder in the LLNA. Further the sensitisation effect is very weak because at 25% with maybe left less observeable irritation effects the SI was only slight above the threshold. But based on the current available data this result differences can not fully explained, therefore RA to the closer homologue with the most detrimental results is proposed.

Migrated from Short description of key information:
Mouse local lymphnode assay (LLNA): sensitising (RL1, GLP, OECD 429, mouse, Harlan 1445505, 2012)

Justification for classification or non-classification

Skin sensitization was observed in a skin sensitization local lymph node assay with the source substancePolyethylene glycol ether with 2-butyne-1,4-diol (CAS 32167-31-0). The scores obtained from the study led to a classification for skin sensitization according to GHS (Regulation (EU) 1272/2008) and to EU-criteria DSD (67/548/EEC).

This classification was also supposed for the target substance2-Butyne-1,4-diol, polymer with methyloxirane (CAS 61596-96-1).

 

Labelling skin/respiratory sensitization:

 

GHS: Category 1b, H317

DSD: R43