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Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: According to ECHA Practical Guide 6 the maximum score for read across is rel. 2

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
A combined reproduction, neonatal development, and neurotoxicity study with 1,6-hexamethylene diisocyanate (HDI) in the rat
Author:
Astroff AB et al
Year:
2000
Bibliographic source:
Reproductive Toxicology 14: 135-146
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): 1,6-hexamethylene diisocyanate (HDI)
- Physical state: liquid
- Purity: 99.6-99.7 %
- Lot/batch No.: 70643
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Portage, MI
- Age at study initiation: 7-9 wks
- Weight at study initiation: Males: 312-383 g; Females: 201-248 g
- Housing: individual
- Diet: ad libitum (except during the exposure period)
- Water: ad libitum
- Acclimation period: at least 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40 - 60
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
TEST SUBSTANCE GENERATION:
HDI was generated as a vapor by passing filtered, dry air through liquid HDI in a grass bubbler. During vapor generation the bubbler containing HDl was immersed in a constant temperature water bath. The vaporized material was entrained with chamber intake air flow for mixing at the chamber head. Both bubbler temperature and air flow may have been adjusted to maintain desired chamber HDl concentrations and these parameters were monitored continuously with recordings at half-hour intervals (minimum) during eaeh six-hour exposure period.

EXPOSURE SYSTEM:
Chambers: The chambers used in this study were Hazleton H-2000 inhalation exposure chambers which are constructed of stainless steel with clear glass windows. Each chamber has an approximate volume of two cubic meters. The chambers are equipped with stainless steel, wire mesh cage-packs. Each cage-pack is fitted with removable feed troughs and an automatic watering system. The air supplied to the chamber passes through an activated charcoal trap and a HEPA filter before being conditioned to the desired temperature and relative humidity. These chambers have been used
previously for exposure of animals to HDI.

NOMINAL CHAMBER PARAMETERS (During Exposure):
Temperature: 22 ± 2°C; Relative Humidity: 50 ± 10%; Exhaust Flow: 700 ± 100 Lpm; Static Pressure: -0.25 to -1.0 inches of water relative to atmospheric.
To the extent possible these nominal values were maintained during each exposure period. During non-exposure periods (nights) nominal values for each chamber parameter were set to be maintained as Iisted above, with the exception that the range for RH shall be relaxed to be 40 - 70%. This was to accommodate expected inereases in RH due to chamber handling and animal care. The increase to 70% RH is in accordanee with AALAC guidelines governing care of rats.
Details on mating procedure:
Mating was accomplished by co-housing one female with one male for up to 15 consecutive days. On each day of the mating phase all animals were exposed to HDl in the inhalation chambers. Following exposure, all animals were transferred into another animal room and co-housed overnight. On each morning following co-housing, but prior to being transferred back to the inhalation chambers, the females were evaluated for evidence of insemination by the presence of sperm in the vaginal smears or an internal vaginal plug. Following this examination all animals were returned to the inhalation chambers for exposure to HDl. Inseminated females remained in the inhalation chambers through gestation day 19 (following exposure on gestation day 19 the inseminated females were transferred to plastic cages. The corresponding male remained in the inhalation chamber through mating day 15 (males were terminated on mating days 16 and 17). The day on which insemination was observed in the vaginal smear was designated day 0 of gestation for that female. Females which did not exhibit sperm in the vaginal smear or an internal vaginal plug were sacrificed following the mating phase and underwent a gross necropsy.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber samples were collected near the animal's breathing zone using two midget impingers connected in series. Samples were collected at a frequency that ensured that the average daily value was representative of the required concentration. At a minimum, three samples (one for the control
chamber) were collected per chamber per day. An acetonitrile solution (at least 10 mL per impinger) containing N-4-nitrobenzyl-N-n-propylamine (nitro reagent) was used to trap and derivatize HDl to a UV-absorbing compound. All midget impinger samples were assayed by an established high performance liquid chromatography method.
Duration of treatment / exposure:
Exposure period: males: 28 days; females: 50 days
Premating exposure period (males and females): 14 days
Duration of test: 54 days
Frequency of treatment:
6 hours/day, 7 days/week
Details on study schedule:
During a 4-day lactation phase the exposure to HDI was also discontinued for the dams. Deviation in the exposure regimen of the females: some of the females were exposed through gestation day 19, others were exposed through gestation day 18 only, and others were exposed through gestation day 18 and again on gestation day 20. In order to assess the impact of the deviation on the study the tissues from the respiratory tract of all females were examined microscopically. The results of these examinations did not indicate any differences between the females differentially exposed.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 0.005, 0.050, 0.300 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 0.005, 0.053, 0.299 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
15
Control animals:
yes, sham-exposed
Details on study design:
DOSE SELECTION RATIONALE:
The concentrations of HDl used in this study were based on a 21-day inhalation toxicity study, a 90day inhalation toxicity study, a chronic inhalation toxicity/oncogenicity study, and a sensory irritation study. In the 21-day study, Sprague-Oawley rats were exposed to either 0, 0.005, 0.0175,
0.15, or 0.3 ppm HDl for 5 hours/day, 5 days/week for 3 weeks. Compound-related ocular and nasal irritation were observed in animals exposed to 0.0175, 0.15, and 0.3 ppm on days of exposure only. These findings were not observed during non-exposure days. There were no compound-related effects on body weight, feed consumption, clinical chemistry, hematology, urinalysis, or gross pathology. At 0.3 ppm, liver and kidney weights were decreased in females. The major findings for both sexes were histopathologie lesions of the nasal mucosa and minor changes in the larynx and trachea. This study demonstrated that the target site following HDl exposure was the nasal cavity. In the 90-day study, Fischer 344 rats were exposed to HDl concentrations of 0, 0.01, 0.04 and 0.14 ppm for 6 hours/day, 5days/week for approximately 13 weeks. The only compound-related findings were ocular irritation and histopathologic lesions of the anterior nasal cavity. Both findings were observed at all three concentrations, therefore, a clear NOEL was not established in this study. In the chronic/oncogenicity study, Fischer 344 rats were exposed to HDl concentrations of 0, 0.005, 0.025 and 0.175 ppm for 6 hours/day, 5 days/week for up to 2 years. Animals were evaluated following both one and two years of exposure. A maximum tolerated dose was achieved at the highest concentration based on decreased body weight and slight anemia in the females, and histopathologie lesions of the nasal cavity in both sexes. The lowest concentration (0.005 ppm) was shown to be a NOEL after one year of exposure. However, after two years of exposure 0.005 ppm was considered to be a NOAEL based on the observation of reversible lesions, indicative of responses to non-specific irritation. In the sensory irritation study, female Sprague-Dawley rats were exposed using the head-only technique, to 0, 0.10, 0.21, 0.79, and 4.42 ppm Mondur HX (100% HDl) for three hours. Following exposure the animals were held for a seven-day recovery period. A concentration dependent increase in the respiratory response (sensory irritation) was observed. The severity of the response culminated in the death of two rats at the 4.42 ppm dose level. The RD50 (concentration which was estimated to produee a 50% depression in respiratory frequency) for the last hour of a three-hour exposure was 1.69 ppm. The NOEL for this study was 0.1 ppm. Based on these results, and the projected exposure of the animals for approximately six weeks during the current study, the proposed concentrations were 0, 0.005, 0.05, and 0.3 ppm HDI.
Positive control:
none

Results and discussion

Results: P0 (first parental animals)

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Remarks:
parental toxicity
Effect level:
0.005 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Microscopic alterations in the nasal cavity at next higher concentration
Dose descriptor:
NOEL
Remarks:
fertility
Effect level:
0.3 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: No effect on fertility up to the highest dose tested

Results: F1 generation

Effect levels (F1)

Dose descriptor:
NOEL
Remarks:
pub toxicity
Generation:
F1
Effect level:
0.3 ppm (nominal)
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

For justification of Read across and Weight of Evidence conclusions see documents attached to the endpoint summary for reproductive toxicity:

- Justification for Grouping and Read across approach....., 2016 -03 -22

- Rationale for Waiving Additional Animal Studies on Reproductive Toxicity ....., 2009 -11 -18

Although studies on fertility, respectively multi-generation studies, are not available for the substance, further testing should be omitted. Weight of evidence conclusions (REACH, Annex XI, 1.2) based on mechanistic toxicity from other structurally related aliphatic isocyanates give evidence to conclude that the substance is not a reproductive toxicant. Consequently, a data waiving is claimed.

Comments for HDI:

Evidence of toxicity was demonstrated in the 0.300 ppm and to a lesser extent in the 0.050 ppm dose group. In the 0.300 ppm dose group a statistically significant decrease in body weight was observed in the females on day 4 of the study. No effects on body weight were observed in the females of the 0.050 or 0.005 ppm dose groups, or the males of any dose group. Also observed at the 0.300 ppm dose level, in both males and females, were microscopic alterations in the nasal cavity, primarily epithelial hyperplasia, squamous metaplasia, chronic-active inflammation, and more seriously, degeneration of the olfactory epithelium. Similar microscopic effects were also observed, albeit to a lesser extent, in the males and females of the 0.050 ppm dose level. No histopathological effects were observed in the 0.005 ppm dose level. There were no statistically significant effects on the mating, fertility, or gestation indices. There were no effects observed on the days to insemination, gestation length, or total number of implantation sites. The NOEL for effects on reproductive parameters was 0.300 ppm. There were no statistically significant effects on litter size, total number of pups born, sex distribution, mean weight of viable pups, mean number of viable pups or number of stillborn pups. No statistically significant effects were observed on the live birth, viability, lactation, or birth indices. The NOEL for effects on litter parameters was 0.300 ppm. 1,6-Hexamethylene diisocyanate demonstrated toxicity at vapor concentrations of 0.050 and 0.300 ppm. No effects were observed in the 0.005 ppm group, and no effects on hematology, clinical chemistry, reproduction (including neonatal development), or neurologic parameters were observed with any concentration. Therefore, the no-observed-effect-level (NOEL) for hematology, clinical chemistry, reproduction, and neurotoxicity for this study was 0.300 ppm and the overall NOEL was 0.005 ppm. Analytically confirmed overall (for the entire study) mean HDI vapour concentrations were 0.005, 0.053 and 0.299 ppm.

Applicant's summary and conclusion

Executive summary:

In a combined reproductive/developmental/neurotoxicity study (OECD TG 422) with 1,6-hexamethylene diisocyanate (HDI) rats were exposed, via whole-body exposure, to HDI vapour concentrations of 0, 0.005, 0.050, or 0.300 ppm for 6 hours/day during a 14-day premating phase, up to a 14-day mating phase, and a 21-day gestation phase. Analytically confirmed overall (for the entire study) mean HDI vapour concentrations were 0.005, 0.053 and 0.299 ppm. Following the gestation phase the dams were transferred to nesting cages and permitted to deliver. The dams and their litters were maintained for a 4-day lactation phase during which exposure to HDI was discontinued. HDI demonstrated toxicity at vapour concentrations of 0.050 and 0.300 ppm resulting in microscopic alterations in the nasal cavity (primarily epithelial hyperplasia, squamous metaplasia, chronic-active inflammation, and more seriously, degeneration of the olfactory epithelium). No effects were observed in the 0.005 ppm group, and no effects on hematology, clinical chemistry, or neurologic parameters were observed with any concentration. There were no statistically significant effects on the mating, fertility, or gestation indices. There were no effects observed on the days to insemination, gestation length, or total number of implantation sites. 

There were no statistically significant effects on litter size, total number of pups born, sex distribution, mean weight of viable pups, mean number of viable pups or number of stillborn pups. No statistically significant effects were observed on the live birth, viability, lactation, or birth indices.

Therefore, the no-observed-effect-level (NOEL) for reproduction (including neonatal development) as well as for hematology, clinical chemistry, and neurotoxicity was 0.300 ppm (2.03 mg/m3) and the overall NOEL was 0.005 ppm (0.034 mg/m3).