Reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-d

Endpoint summary

Description of key information

The key study is an LLNA on the reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-diol. Supporting studies are available in humans and guinea pigs for triethylene glycol (TEG) and tetraethylene glycol (TTEG), components of the reaction mass.

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:

June 16-June 28, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Test System : Mice
Species (strain) : Mus musculus (CBA/J)
Animal Source : Animal Breeding Facility, Jai Research Foundation
Number of Animals Used : 8 (2 mice/group) for screening study and 25 (5 mice/group) for main study
Sex : Females (nulliparous and non- pregnant)
Initial Body Weight (g) On Day 1 (Main Study) : Minimum: 18.7, Maximum: 26.2
Age (Main Study) : 11 to 12 weeks old at the initiation of treatment

Mice were received into the experimental procedure room after veterinary examination for health condition and allowed to acclimatise to the laboratory conditions for a period of 6 days prior to commencement of dosing.
Caging : Mice were housed individually in solid floor polypropylene (size: 290 mm x 220 mm x 140 mm) solid bottom cages which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). These cages had stainless steel top grills through which pellet feed and drinking water were provided; steam sterilized corn cob bedding was used and changed along with the cage at least twice a week. On test day 6, animals were housed in metabolic cages. Cage was rotated once in a week.
Water Bottle : Each cage was supplied with a polypropylene water bottle (capacity 300 mL) with a stainless steel nozzle.
Enrichment : Mice were provided with tunnels in each cage.
The quality of feed and water is regularly monitored at Jai Research Foundation; copies of the relevant certificates of analysis are kept in the study file. There were no known contaminants in the feed and water at levels that would have interfered with the experimental results obtained.
Diet : Teklad Certified Global High Fiber Rat/Mice feed manufactured by Envigo, USA, was provided ad libitum.
Water : UV sterilised water (Reverse Osmosis water filtration system) was provided ad libitum.
Environmental Conditions
Animal Room : BMR 30 [On test day 6, all animals were shifted to Room N° 414]
Temperature Range : 20 to 23 °C
Relative Humidity Range : 57 to 66%
Photoperiod : The photoperiod was 12 h artificial light and 12 h darkness, light hours being 06:00 – 18:00 h (photoperiod maintained through automatic timer).
Air Changes Rate : 16 air changes/hour
Randomization
After acclimatisation animals were randomized into different groups using in-house developed,
validated computer software.

Vehicle:

dimethylformamide

Concentration:

10%, 50% (v/v), 100%

No. of animals per dose:

Screening study: 2 mice/group
Main study: 5 mice/group

Details on study design:

Solubility Test
Prior to the irritation screening test, a solubility test was conducted to identify an appropriate vehicle for testing Pentaethylene Glycol Mixture. The test item was identified to be not soluble in guideline recommended LLNA vehicle, acetone/olive oil (4:1 v/v at concentrations above 25% (v/v)). Pentaethylene Glycol Mixture formed a uniform solution at 75% and lower concentrations in N,Ndimethylformamide (DMF) as a vehicle. Therefore, following consultation with the Sponsor, DMF was selected as the vehicle.

Screening Test
An irritation screening test was conducted to identify the highest dose that does not cause irritation or overt systemic toxicity. The study consisted of four groups of mice (2 mice per group) that were treated with Pentaethylene Glycol Mixture at concentrations of 25%, 50%, 75% (v/v) in DMF and 100% (undiluted, as received) (25 μL/ear) for three consecutive days (days 1, 2 and 3).

Dose concentrations of 25%, 50%, 75% (v/v) in DMF and 100% (undiluted, as received) of Pentaethylene Glycol Mixture% was selected based on maximum solubility, compatibility, and viscosity of the test item with the vehicle that could be applied to the mouse ear (documented in the raw data). All mice appeared active and healthy, and there were no consistent treatment-related effects on body weight. There was no dermal irritation and no increases in ear swelling of 25% or greater observed in the 25%, 50%, 75%, or 100% test concentration groups.

The Local Lymph Node Assay
Prior to treatment, the animals were weighed and the ears were checked for any abnormalities or clinical signs of diseases or injury. Twenty-five healthy naive female mice without pre-existing ear irritation were selected and distributed into treatment groups (5 mice per group). Three treatment groups (G12 to G14) were treated topically once daily for three consecutive days (days 1, 2 and 3) on the dorsal surface of both ears (25 μL/ear) using a calibrated micropipette with Pentaethylene Glycol Mixture at concentrations of 10%, 50% (v/v) and 100%, respectively. The dose was gently spread evenly over the dorsal surface of the ear using the tip of the pipette. Mice from the vehicle control group (G11) and positive control group (G15) were handled in the same manner but received 25 μL/ear of vehicle (DMF) or 25% α-Hexylcinnamaldehyde (v/v) in vehicle (DMF), respectively. No treatment was applied on days 4 and 5 for any group. All dosage preparations were freshly prepared on the day of application.

On day 6 (approximately 72 h after the last treatment), all mice from the vehicle control, positive control and all treatment groups were intravenously injected via the tail vein with 250 μL of sterile phosphate buffered saline (PBS) containing approximately 20 ± 1 μCi (740 KBq) of 3H-methyl thymidine.

Body weights were recorded on day 1 and prior to thymidine administration on day 6. Animals were observed for clinical signs, local irritation at the site of application, and systemic toxicity. Local irritation responses were made as per criteria given in OECD 429, 2010; Section 22.

Lymph nodes were collected on day 6, 5 hours after thymidine administration.

Incorporation of 3H-methyl thymidine was measured by β-scintillation counting as DPM for each mouse and expressed as DPM/mouse. The total radioactivity of each sample was counted using LSA (Liquid Scintillation Analyser) after 30 minutes of mixing with scintillation fluid and required quench corrections were made. The quench curve was established using extended quench standards (PerkinElmer) of 253100 ± 1.6 DPM β s ource ( July 2 6, 2 012). Computer constructed quench curve was derived from the above commercially available series of scaled standards which automatically converts Counts Per Minute (CPM) to DPM.

The proliferate response of lymph nodes from each mouse was expressed as the number of radioactive DPM per mouse, calculated by subtracting out background DPM (measured in 1 mL of 5% TCA aliquot).

SI = mean DPM of test group divided by mean DPM of solvent/vehicle control group
Any test item that produces a SI > 3 in the LLNA is considered “positive” for dermal sensitization potential (Kimber et al., 1994).
Based on the EC3 values derived from the LLNA, it has been proposed that contact allergens can be categorized as weak (> 10% - < 100%), moderate (> 1% - < 10%), strong (> 0.1% - < 1%), or extreme (< 0.1%) (ECETOC, 2003).

Positive control substance(s):

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

Statistics:

The DPM/mouse, along with an appropriate measure of inter-animal variability (i.e., mean ± standard deviation), were calculated for each test group and vehicle and positive control groups. Final results were expressed as the (SI) which is calculated as a ratio of the mean DPM of test group divided by mean DPM of vehicle control group.

EC3 value (theoretical concentration resulting in a SI value of 3) is determined by linear interpolation of points on the dose-response curve, immediately above and below the 3-fold threshold (Basketter et al., 1999). The equation used for calculation of EC3 was: EC3 = c + [(3 - d)/(b - d)] x (a - c), Where a = the lowest concentration giving stimulation index > 3; b = the actual stimulation index caused by a; c = the highest concentration failing to produce a stimulation index of 3; and d = the actual stimulation index caused by c.

In addition to an assessment of the magnitude of the SI, statistical analysis was carried out for the assessment of the dose response relationship and pair-wise comparison made between the treatment and the solvent/vehicle control group. All the parameters characterised by continuous data such as body weight and radioactive disintegrations per minute (DPM) were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA). To compare vehicle and positive control data, Student's t-test was performed to calculate significance.

Parameter:

SI

Value:

1.65

Test group / Remarks:

10% Pentaethylene Glycol Mixture in DMF

Parameter:

SI

Value:

2.16

Test group / Remarks:

50% (v/v) Pentaethylene Glycol Mixture in DMF

Parameter:

SI

Value:

2.75

Test group / Remarks:

100% Pentaethylene Glycol Mixture

Parameter:

SI

Value:

6.02

Test group / Remarks:

25% HCA (positive control)

Cellular proliferation data / Observations:

Proliferative responses in the draining lymph nodes were monitored by measuring the incorporation of 3H-methyl thymidine. These analyses revealed group mean DPM values of 1288.80, 2131.40, 2790.20 and 3546.60 for the vehicle control (DMF), 10%, 50% (v/v) in DMF and 100% dose concentrations of Pentaethylene Glycol Mixture, respectively. The DPM value for the positive control (25% α-Hexylcinnamaldehyde) was found to be 7752.40. The DPM values observed in all the groups treated with Pentaethylene Glycol Mixture and the 25% HCA treated group were statistically significant when compared to control values.

Stimulation Index (SI) values calculated for groups treated with Pentaethylene Glycol Mixture were found to be 1.65, 2.16 and 2.75 at the dose concentration of 10%, 50% (v/v) in DMF and 100%, respectively, and 6.02 for the 25% HCA treated positive control group. The SI obtained for Pentaethylene Glycol Mixture at 10%, 50% (v/v) in DMF and 100% showed less than three-fold increase over the control value, an EC3 calculation was not possible.

Pentaethylene Glycol Mixture did not demonstrate dermal sensitisation potential in the local lymph node assay.

Table 1: Summary of DPM and SI Value

Dose Concentration (%)	# of Mice Used	Group Mean DPM	Standard Deviation	Stimulation Index (SI)
Vehicle control DMF	5	1288.80	320.37	1
10% Pentaethylene Glycol Mixture	5	2131.40*	552.18	1.65
50% Pentaethylene Glycol Mixture	5	2790.20**	555.34	2.16
100% Pentaethylene Glycol Mixture	5	3546.60**	305.95	2.75
Positive control 25% HCA	5	7752.40**	1851.66	6.02

* = Significantly higher than control (p ≤ 0.05)

** = Significantly higher than control (p ≤ 0.01)

No clinical signs observed in any group. No dermal irritation in Pentaethylene Glycol Mixture treated animals; very slight erythema observed in days 2 and 5 in all mice of the positive control group. The mean body weight of positive control as well as Pentaethylene Glycol Mixture treated mice was comparable to that of the control group.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study, Pentaethylene Glycol Mixture is considered negative for dermal sensitization potential in the LLNA.
Proper conduct of the LLNA was confirmed via a positive response with 25% α-Hexylcinnamaldehyde (HCA), a weak contact sensitizer.

Executive summary:

This study was conducted to examine the dermal sensitisation potential of Pentaethylene Glycol Mixture using the mouse local lymph node assay (LLNA).

A preliminary assay was conducted to identify the appropriate test concentrations for the main study.

Based on the results from a preliminary assay, five groups of mice (each comprising 5 females) were selected for the experiment. Three groups were treated with Pentaethylene Glycol Mixture at concentrations of 10%, 50% (v/v) in N,N-dimethylformamide (DMF) and 100% for three consecutive days (days 1, 2 and 3) on the dorsum of both ears (25 μL per ear). One group served as a vehicle control and was treated with DMF, and another group served as a positive control and was treated with α-hexylcinnamaldehyde (HCA) at a concentration of 25% (v/v) in DMF.

Group mean body weights of treated animals were comparable with the control group and there were no indications of clinical or systemic toxicity in Pentaethylene Glycol Mixture treated animals.

On day 6, the uptake of 3H-methyl thymidine into the auricular (local) lymph nodes draining at the site of chemical application was measured (5 hours post 20 ± 1 μCi intravenous (i.v.) injection) to assess the lymph node proliferative response. All animals were euthanized via CO2 asphyxiation and the lymph nodes were harvested and prepared for analysis in a scintillation counter. The results are presented in Disintegrations Per Minute per mouse (DPM/mouse). Each animal’s ears were also evaluated for erythema and edema prior to each application and again on days 4 and 5 and on day 6, prior to the i.v. injection. A positive response for HCA (Stimulation Index; SI = 6.02) confirmed the reliability of the test procedure. Mean stimulation indices for the 10%, 50% (v/v) and 100% Pentaethylene Glycol Mixture treated groups were 1.65, 2.16 and 2.75, respectively (i.e., less than three-fold increase over mean control group). Therefore, Pentaethylene Glycol Mixture did not demonstrate dermal sensitisation potential in the local lymph node assay. All criteria for a valid study were met as described in the protocol. The vehicle control and positive control in the definitive LLNA were within the acceptable ranges and fulfilled the requirements for a valid assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The key study is a local lymph node assay (LLNA) assessing sensitization potential of the reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-diol (JRF, 2016). Female mice were treated with concentrations of 10, 50, or 100% for 3 days on the dorsum of both ears and the lymph node proliferative response was assessed on day 6. Mean stimulation indices of 1.65, 2.16, and 2.75 for the 10, 50, and 100% concentrations of the mixture demonstrated no sensitization potential.

This is supported by additional evidence from supporting studies conducted on two of the individual components of the registered substance by TKL Research, Inc. (1989). In this repeated human patch test study, TEG or TTEG was applied (0.2 mL) semiocclusively or occlusively to the skin of 397 individuals every 48 hours for 9 applications. Subjects removed the patches after 24 hours. Following a two week rest phase, subjects received a challenge exposure to a previously unexposed site for 24 hours. Application sites were graded at 48 and 72 hours; no evidence of human sensitization was observed for TEG or TTEG.

The sensitization potential of TEG and TTEG was also examined in two supporting studies using the Magnusson and Kligman method in guinea pigs (Biodynamics Inc., 1990a, 1990b). No sensitization potential was observed with TEG or TTEG in guinea pigs.

Based on these data, the reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-diol is not expected to produce dermal sensitization.

Respiratory sensitisation

Endpoint conclusion

Additional information:

Based on low irritation potential and negative dermal sensitization data, respiratory sensitization from exposure to the reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-diol is not expected to occur.

Justification for classification or non-classification

A guideline compliant LLNA is available for the reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-diol is available. Negative results for sensitization potential in the LLNA, as well as negative results in human repeat patch testing on the components TEG and TTEG, demonstrate that classification is not warranted.