Distillation residue, butyl alcohols production, rectification

Endpoint summary

Description of key information

Skin irritation/corrosion in vitro: non-corrosive.
Skin irritation/corrosion in vitro: irritating, read-across information from surrogate (2-ethylhexanol). 
Eye irritation in vitro: irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

1992-03-06

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Repeated-dose dermal toxicity, similar to that of OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test), studies performed with the only identified and quantified constituent of the registered substance, 2-ethylhexanol. Studies performed with pregnant Fischer F344 rats and they study the developmental toxicity of 2-ethylhexanol. In the absence of the registered substance these studies serve as a good surrogate studies: a) it represents the effects of the only identified and quantified constituent of the substance, since the constituents of this registered UVCB-substance cannot be quantified and its composition varies to the degree that composition cannot be fixed. b) studies have been performed with good quality and data is published in a peer-reviewed journal. c) they provide adequate information on effect levels of 2-ethylhexanol when administered via dermal route.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD TG422: dermal route

Deviations:

yes

Principles of method if other than guideline:

Pregnant Fischer 344 rats were studied in two studies with an occluded dermal application:
1. Range-finding study
2. Main study

GLP compliance:

yes

Remarks:

U.S EPA Health effects guidelines and Good Laboratory Practice regulations.

Species:

rat

Strain:

Fischer 344

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Virgin F344 rats (CDF(R) F344 Crl./Br.)
- Source: Charles River Breeding Laboratories (Kingston, NY)
- Age at study initiation: 70 days / 63 days
- Weight at study initiation: males 175-200g / females 130-150g
- Gestational day 0: appearance of copulatory plug
- Housing: singly
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2-week quarantine period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 42-65
- Air changes (per hr): not disclosed
- Photoperiod (hrs dark / hrs light): 12 hour

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

other: positive control for adverse effects on reproduction: 2-methoxyethanol

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
1.Rangefinding study: 0.5, 1, 2, 3 (ml/kg/day) equivalent to 420, 840, 1680, 2520 (mg/kg/day). Positive control: 2-methoxyethanol: 0.5 and 1.5 (ml/kg/day) equivalent to 420 and 1260 (mg/kg/day). Oral cavage reference compound: Valproic acid 400 mg/kg bw/day.
2. Main study: 0.3, 1, 3 (ml/kg/day) equivalent to 252, 840, 2520 (mg/kg/day).Positive control: 2-methoxyethanol 1 (ml/kg/day) equivalent to 840 (mg/kg/day)
- Concentration (if solution): 100%
- Constant volume or concentration used: no


VEHICLE
- No vehicle

Duration of treatment / exposure:

6-hours day

Observation period:

Before and after treatment

Number of animals:

Range-finding study: 8 animals per group
Main study: 25 animals per group

Details on study design:

TEST SITE
- Area of exposure: clipped and shaved dorsal skin of 9,7 cm2 (in the report ca. 1,5 cupic inches)
- Type of wrap if used:2 cupic inch gauze square, occluded with a Lycra-Spandex with Velcro closures. A 1.5x2.5 in. polyethylene patch was attached at the application site under the jacket.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): wiped gently with moist gauze and blotted dry.
- Time after start of exposure: 6-hours


SCORING SYSTEM:
Draize score, FHSA (1987)

Irritation parameter:

overall irritation score

Basis:

mean

Remarks:

Draize score

Time point:

other: before and after 6-hour treatment

Score:

< 1.1

Max. score:

1.1

Reversibility:

fully reversible within: 18 hours

Remarks on result:

other: effects occurred during treatment and were transient or ameliorated after treatment ceased

Irritant / corrosive response data:

DERMAL IRRITATION
Treatment -related effects attributable to 2-ethylhexanol at the application site were exfoliation, encrustation and erythema. There was no edema. Exfoliation and encrustation occurred in both range-finding and main studies at all treatment levels of 2-ethylhexanol. There was no erythema or edema in sham controls. Erythema occurred during treatment with 2-ethylhexanol at levels of 830 mg/kg bw/day and above. Draize scores revealed that irritation was essentially mild. Maximum mean treatment scores occurred on gd10 at 1680 mg/kg bw/day (draize-score 0.4, range-finding study), on gd 11 at 2520 mg/kg bw/day (draize-score 1.1, range-finding study), and on gd 14 at 1680 mg/kg bw/day (draize-score 0.3, main study).

There was no exacerbation by continuent treatment. Erythema subsided immediately after cessation of treatment. There was no exfoliation, encrustation, erythema or edema at the application of control, 2-methoxyethanol.

Nasal encrustation and ocula encrustation and discharge were seen mostly in the main study with 2-ethylhexanol and 2-methoxyethanol and in sham controls. Since these effects occurred in controls and mostly disappeared after treatment ceased they are attributed to handling stress.

Other effects:

Weight reduction at highest concentrations (1680 and 2520 mg/kg bw/day).

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Repeated dermal exposure of 2-ethylhexanol lead with high doses (1680 and 2520 mg/kg bw/day) to slight reduction in body weight, otherwise it did not indicate properties which would lead to a concern for toxicity via dermal route. There was no exacerbation by continuent treatment. Erythema subsided after cessation of treatment. The only effect was mild skin irritation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-07-27 TO 2010-08-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed following GLP and performed following the Draft Standard Operating Procedure of MatTek Corporation -Eye Irritation Validation Study (EIVS): Ocular Irritation Assay for Chemicals using EpiOcular (tm).

Qualifier:

according to guideline

Guideline:

other:

Deviations:

not applicable

Principles of method if other than guideline:

Study was performed following GLP and performed following the Draft Standard Operating Procedure of MatTek Corporation -Eye Irritation Validation Study (EIVS): Ocular Irritation Assay for Chemicals using EpiOcular (tm).

GLP compliance:

yes (incl. QA statement)

Remarks:

Hameln rds a.s., Section of Biological studies, Horna 36, 900 01 Modra, Slovak Republic.

Species:

other: human corneal model

Strain:

not specified

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

Amount / concentration applied:

50 microliters topically applied

Details on study design:

Test system
specific model for eye irritation testing EpiOcularTM (OCL-200) (Lol No. 13059) was obtained from MatTek Corporation. USA. OeL-200 kit was supplied by the MatTek Corporation instead of ordered OCL-21 2 kit (12 tissues). The difference between these two kits is only in the number of tissues.

Control material
Concurrent negative and positive controls were used in experiment to ensure adequate performance or lhe experimental model.
Negative control (NC); Sterile deionised water
Positive control (PC); Ethyl acetate, p.a.

Chemicals, media
Assay Medium OCL- IOO·ASY (Dulbecco's Modified Eagles medium - DMEM) Lot 072310A IIB (MatTek). Phosphate Buffered Saline (PBS) Lot 28104117 (Flow Laboratories). Elhyl acetate p.a. Lot. 1523872008 (Merck). MTT (3-[4 ,5-Dimethylhiazol·2·yI]-2 ,5-diphenyltetrazolium bromide) 98% Lot. 51K5312 (Sigma). isopropanol p.a. Lot. A090313/01 (Mikrochem), sterile deionized Water, tissue culture grade (hameln rds a.s.).

Protocol
In vitro eye irritation: specific human corneal model test was used. Test was performed in compliance with Draft Standard Operating Procedure of MatTek Corporation - Eye Irritation Validation Study (EIVS): Ocular Irritation Assay for Chemicals using EpiOcular (tm) and in compliance with Principle GLP Section of Biological Studies (2008). Two tissue replicates were used for each treatment (exposure time), including controls. Protocol for liquid test articles was used: test product was tested by applying of 50 microliters topically on cultures for 30 minutes, followed by a 12-minute post-trealment immersion and a 120-minute post-treatment incubation. The tissues were incubated at standard culture conditions (37 ± 1°C) in a humidified atmosphere of 5 ± 1 % C02 in air). After exposure period (30-min). each tissue will be rinsed gently with PBS to remove any residual test article. The magnitude of viability was quantified by using MTT test (SOP I).

Test article treatment
After the 30-minute pre-treatment with PBS. test product (concentrated) was dispensed directly atop the EpiOcular™ tissue at volume of 50 microliters. After exposure period (30-min), each tissue was rinsed extensively with PBS to remove any residual test article (three clean beakers containing 100 mL each of PBS were used).

Test for direct MTT reduction
A test substance may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present in the tissues when the MTT viability test is performed. To identify this possible interference of test product. this test was performed in the previous study (4).

Receipt and preparation of cultures
EpiOcularTM was delivered one day prior or experiment performing. Each culture was removed with sterile forceps from the agarose gel, inspected, and transferred to a pre-labeled 6-well plates containing 1 mL of assay medium per well. The EpiOcular™ cultures were incubated at 37 ± 1°C in a humidified atmosphere of 5 ± I % C02 in air for overnight prior to dosing for release of transport stress related compounds and debris.

Assay procedure
After overnight pre-incubation tissues were pre-treated with 20 mL of PBS without calcium and magnesium. The tissues were incubated at standard culture condition for 30 minutes. Then, 50 microliters of the test or control articles were applied topically onto the tissue surface for 30 minutes. Two tissues each were used per treatment, negative and positive control. The cultures were returned to the incubator for the appropriate exposure times. After this, the test and control articles were extensively rinsed from the cultures using PBS without calcium and magnesium. After rinsing, the tissues were immediately transferred to and immersed in 5 mL of pre-warmed Assay medium in a prelabelled 12-well plate for a 12 min immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion, each insert was transferred to the appropriate well of the prelabelled 6-well plate containing 1 mL of Assay medium. The tissues were incubated for 120 min at standard culture conditions (Post-treatment incubation). After this, the cultures were transferred to 24-wells plate containing 0.3 mL/well of MTT reagent (1 mg/mL) and incubated at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air for 3 hours.After incubation, the cultures were blotted on absorbent paper and extracted in 2 mL of isopropanol overnight without shaking at room temperature. At the end of the extraction period, the liquid with in each insert was decanted into the well from which it was taken. The extract solution was mixed for 15 min and two 200 microliters aliquots were transferred to a 96-well plate and the absorbances were recorded.

Treatment of results
Data are included cytotoxicity and viability determination. The optical densities (ODs) were read in a 96-well plate spectrophotometer using a wavelength 540nm without a reference filter. Data of optical densities generated by microplate reader and already corrected by substraction of the blanks mean from all OD values were manually transported into the first map (Import) of the EXCEL spreadsheet in the 96-well format. The second sheet (Results) were made calculations and provided a column graph of the results. The OD values obtained for each test sample were used to calculate a percentage viability relative to negative control. which is arbitrarily set at 100 %. All data were summarised in tabular form in protocol (1, 2).

Assay acceptance criteria
Negative control:
The absolute optical density (OD) of the H2O treated NC tissues in the MTT-test is an indicator of tissue viability obtained under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the mean of NC is OD>= 1.
Positive control:
30-min exposure of the PC must reveal a mean tissue viability less than 60 %.

Evaluation criteria
The prediction of eye irritation associated with EpiOcular™ model is:
• If the test article-treated tissue viability is > 60% relative to negative control-treated tissue viability, the test article is labeled non-irritant.
• If the test article-treated tissue viability is < 60% relative to negative control-treated tissue viability, the test article is labeled irritant

Irritation parameter:

other: MTT reduction score

Basis:

mean

Time point:

other: 120 minutes

Score:

>= 27 - < 28.8

Max. score:

100

Reversibility:

not specified

Remarks on result:

other: Threshold is 60%, if below than considered as irritant to eye

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The substance was examined for in vitro eye irritation in human corneal model test EpiOcular(tm). Validity of the test method was ascertained by positive control ethyl acetate. Two tissue replicates were used for each treatment (exposure time 30-min, including controls). The magnitude of viability was quantified by using MTT-test. Determined viability of culture treated with substance fulfilled criteria for eye irritation test. It has been concluded that substance is
considered to be irritant to eye.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation

The weight of evidence approach is used to determine the skin irritation of this substance . There is one published study (Tyl, et al. 1992) available on 2-ethylhexanol to evaluate the skin irritation property. 2-ethylhexanol is the only identified and quantified constituent of the target substance. This study is similar to that of OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test). The study is performed with pregnant Fischer F344 rats via dermal route. Treatment -related effects attributable to 2-ethylhexanol at the application site were exfoliation, encrustation and erythema. There was no edema. Effects occurred during treatment were transient or ameliorated after treatment ceased.

Furthermore, the target substance was tested in in vitro skin corrosion test, EU method B.40, (Hameln rds a.s., 2010c) and did not indicate corrosive properties.

As a conclusion, the weight of evidence of the skin irritation studies on the target substance and on the 2-ethylhexanol is considered reliable. 2-ethylhexanol is the only identified and quantified constituent of the target substance (> 16 - < 35 % (w/w) 2-ethylhexanol). LEAD registrant of 2-ethylhexanol has proposed classification of Skin Irrit. 2. Based on the mixture rules (GCL >- 10 %) and the proposed classification of 2-ethylhexanol, the target substance will be classified as Skin Irrit. 2.

Eye irritation

There is reliable in vitro eye irritation study conducted for the target substance. The target substance was examined for in vitro eye irritation in human corneal model test EpiOcular(tm). Validity of the test method was ascertained by positive control ethyl acetate. Two tissue replicates were used for each treatment (exposure time 30-min, including controls). The magnitude of viability was quantified by using MTT-test. Determined viability of culture treated with substance fulfilled criteria for eye irritation test. It has been concluded that the target substance is considered to be irritant to eyes.

Respiratory irritation

Irritating effects of 2 -ethylhexanol (2 -EH) on eyes and the respiratory tract were investigated in controlled experimental human studies. In these studies by Kiesswetter et al. (2005) and van Thriel et al. (2005) volunteers were exposed for 4 h to mean exposure levels of 1.5, 10 and 20 ppm. The studies revealed strong dose–response relationships between airborne 2-EH concentrations and blink rates and nasal irritation. In the course of 4 h exposure, blink rates increased significantly showing no adaptation and nasal irritation remained unchanged across.

2-ethylhexanol is the only identified and quantified constituent of the target substance (> 16 - < 35 % (w/w) 2-ethylhexanol). This alcohol causes irritation of the respiratory tract during 4-hour exposure, and has been proposed the classification to hazard class STOT SE 3 H335. Since the concentration of the 2-ethylhexanol in the target substance exceeds the generic concentration limit (GCL > 20 %), the target substance will be classified for STOT SE 3 H335.

Justification for selection of skin irritation / corrosion endpoint:
There is no in vivo study conducted for the target substance. This in vivo study is conducted for the only identifiable and quantified component, 2-ethylhexanol.

Justification for selection of eye irritation endpoint:
Reliable in vitro study available for the target substance.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Effects on respiratory irritation: irritating

Justification for classification or non-classification

Based on the skin irritation information, the substance has to be classified to hazard class Skin Irrit. 2 according to CLP Regulation 1272/2008 and as Xi; R38 according to Directive 67/548/EEC.

Based on the eye irritation information, the substance has to be classified to hazard class Eye Irrit. 2 according to CLP Regulation 1272/2008 and as Xi; R36 according to Directive 67/548/EEC.

The target substance may contain 2 -ethylhexanol up to 35 % (w/w). As 2 -ethylhexanol has been shown to cause irritation of the respiratory tract in the experimental human studies, the substance has to be classified to hazard class STOT SE 3H335 according to CLP Regulation 1272/2008 and as Xi; R37 according to Directive 67/548/EEC.