4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline)

Endpoint summary

Description of key information

Oral (OECD 401), rat: LD50 >5000 mg/kg bw
Inhalation (OECD 436), rat: LC50 >3 mg/L air (maximum attainable concentration)
Dermal (OECD 402), rat: LD50 >2000 mg/kg bw

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Dec - 31 Dec 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Restriction: purity of test substance not specified

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

adopted 12. May, 1981

Deviations:

yes

Remarks:

purity of test substance not given

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI, USA
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 240 - 299 g
- Fasting period before study: 17 to 20 hours (over night)
- Housing: separated by sex in groups of 5 in screen-bottom stainless steel cages (heavy gauge)
- Diet: Purina Rodent Chow, ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 21 - 31
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 Dec To 31 Dec 1987

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 0.5 g/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Justification for choice of vehicle: vehicle was chosen due to good solubility of test substance in this vehicle

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

Doses:

5000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Each animal was observed for clinical signs and mortality at 1, 2.5 and 4 hours following administration, and daily thereafter for 14 days, once daily for clinical signs and twice daily for mortality, weighing was done just before dosing, on day 7 and at study termination
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Statistics:

Average body weights were calculated.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortalities were observed during the study period.

Clinical signs:

other: 1 male animal had diarrhea 4 hours after administration. 2 male animals had miosis starting at 2.5 hours following administration until day 1 and day 2, respectively. Some female animals showed miosis (1, 2.5 and 4 hours after administration and on day 1)

Gross pathology:

One female animal had multiple red pinpoint foci in the right salivary gland and diffusely dark red submandibular bilateral lymph nodes. A second female had an enlarged pelvis in the right kidney. The findings in the two female rats were considered to be incidental and unrelated to exposure. All other animals were without visible lesions.

Table 1: Clinical Signs (Number of animals affected)

	Hours			Days
	1.0	2.5	4.0	1	...	14
Males (5.0 g/kg)
Appeared Normal	5	3	2	4	...	5
Diarrhea	0	0	1	0	...	0
Miosis	0	2	2	1	...	0
Females (5.0 g/kg)
Appeared Normal	2	0	0	4	...	5
Miosis	2	3	3	1	...	0
Diarrhea	2	3	4	0	...	0
Hypoactivity	0	1	2	0	...	0
Lacrimation	0	3	3	0	...	0
Hypersensitivity to touch	0	0	1	0	...	0

Table 2: Average body weights (g)

	Dose Level	Average Body Weights (g)
	(g/kg)	Initial	Day7	Terminal
Male	5.0	280	344	367
Female	5.0	253	279	288

Interpretation of results:

GHS criteria not met

Conclusions:

The LD50 was determined to be > 5000 mg/kg for male and female rats.

Executive summary:

The acute oral toxicity of the test substance to rats was investigated according to OECD TG 401 and following GLP principles. A limit test using a dose of 5000 mg/kg was conducted.

No mortality was observed in males and females.

The oral LD50 value of the test substance was established to be > 5000 mg/kg body weight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 2), and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.5, of Regulation (EC) No 1907/2006.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 - 24 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also in accordance with GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Young adult animals (approximately 10 weeks old) were selected.
- Weight at study initiation: Body weight variation did not exceed +/- 20% of the sex mean.
- Housing: Group housing of three animals per sex per cage in labelled Makrolon cages (type IV; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) except during exposure to the test substance.
- Water (e.g. ad libitum): Free access to tap water except during exposure to the test substance.
- Acclimatization period: at least 5 days before start of treatment under laboratory conditions.
- Health inspection: A health inspection was performed prior to commencement of treatment, to ensure that the animals were in a good state of health.

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: 10 - 24 November 2014

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remark on MMAD/GSD:

The MMAD and GSD were determined twice during the exposure period. The MMAD was 3.2 µm (GSD 2.1) and 3.9 µm (GSD 2.3).

Details on inhalation exposure:

The design of the exposure chamber is based on the flow past nose-only inhalation chamber (Am. Ind. Hyg Assoc. J. 44(12): 923-928, 1983). The chamber consisted of three animal sections with eight animal ports each. Each animal port had its own atmosphere inlet and exhaust outlet. The animals were placed in restraining tubes and connected to the animal ports. The number of animal sections and number of open inlets were adapted to the air flow in such a way that at each animal port the theoretical air flow was at least 1 L/min, which ensures an adequate oxygen supply to the animals. The main inlet of the test atmosphere was located at the top section and the main outlet was located at the bottom section. The direction of the flow of the test atmosphere guaranteed a freshly generated atmosphere for each individual animal.
All components of the exposure chamber in contact with the test material were made of stainless steel, glass, rubber or plastic. To avoid exposure of the personnel and contamination of the laboratory the exposure chamber was placed in a fume hood, which maintained at a slight negative pressure.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Nominal: 44 mg/L
Measured time weighted mean concentration: 3 mg/L


The actual time weighted mean concentration was 3 ± 0.1 mg/L. The nominal concentration (amount of test substance used divided by the volume of pressurized air used) was 44 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 8%.

The concentration measurements (n=15) equally distributed over time showed that it was difficult to maintain a stable level at this technically maximum attainable concentration. The generation was interrupted in order to remove test substance deposits from the system at four occasions with a total interruption time of approximately 30 minutes. The generation time was elongated (total generation time 253 minutes) to compensate for these interruptions, resulting in an actual exposure time of at least 227 minutes. Since the interruptions and variations in concentration were taken into account by calculation a time weighted mean concentration, it was considered that the results of this study are representative for a single exposure of 3 mg/L for 4 hours. This was the technically maximum attainable concentration.

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

Test substance preparation: The test substance was used as delivered by the sponsor.

The test substance was administered as an aerosol by inhalation for 4 hours to two groups of three male and three female Wistar rats each. Animals were subjected to daily observations and determination of body weights on Days 1, 2, 4, 8 and 15 and at death. Macroscopic examination was performed on the day of death or after terminal sacrifice (day 15).

Statistics:

No statistical analysis was performed.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 3 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: Maximum obtainable concentration

Mortality:

No mortality occurred.

Clinical signs:

other: Rapid breathing was seen for the animals during exposure. No clinical signs were noted following the exposure.

Body weight:

Body weight loss was seen for all females during the first week post-exposure (up to Day 8). Two females regained weight during the second week resulting in an overall body weight gain during the complete observation period, while one female remained below the initial body weight at the start of the study. Overall body weight gain in males was within the range expected for rats of this strain and age used in this type of study.

Gross pathology:

No abnormalities were found at macroscopic post mortem examination of the animals.

Interpretation of results:

GHS criteria not met

Conclusions:

The 4h-LC50 of 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) in Wistar rats was established to exceed the maximum obtainable concentration of 3 mg/L.

Executive summary:

The acute inhalation toxicity of the test substance was investigated according to OECD TG 436 and GLP principles. The substance was administered as an aerosol by nose-only inhalation for 4 hours to one group of three male and three female rats. Animals were subjected to daily observations and determination of body weights on Days 1, 2, 4, 8 and 15. Macroscopic examination was performed after terminal sacrifice.

The actual time weighted mean concentration was 3 ± 0.1 mg/L. The nominal concentration (amount of test substance used divided by the volume of pressurized air used) was 44 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 8%. The concentration measurements (n=15) equally distributed over time showed that it was difficult to maintain a stable level at this technically maximum attainable concentration. The generation was interrupted in order to remove test substance deposits from the system at four occasions with a total interruption time of approximately 30 minutes. The generation time was elongated (total generation time 253 minutes) to compensate for these interruptions, resulting in an actual exposure time of at least 227 minutes. Since the interruptions and variations in concentration were taken into account by calculation a time weighted mean concentration, it was considered that the results of this study are representative for a single exposure of 3 mg/L for 4 hours. This was the technically maximum attainable concentration.

MMAOD was measured twice, 3.2 µm (GSD 2.1) and 3.9 µm (GSD 2.3).

No mortality occurred and no clinical signs were noted following the exposure. Rapid breathing was seen during exposure. Overall body weight gain in males was considered normal and after an initial body weight loss in females the first week post-exposure, two out of the three females regained the weight during the second week. No abnormalities were found at macroscopic post mortem examination.

The 4h-LC50 of the test material in rats was established to exceed the maximum obtainable concentration of 3 mg/L

Based on these results 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) does not have to be classified and has no obligatory labelling requirement for acute inhalation toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.5, of Regulation (EC) No 1907/2006.

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 Dec - 17 Dec 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also in accordance with GLP principles.

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Version / remarks:

adopted 1984

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Crj: CD(SD)

Remarks:

Crl. (CD) SD BR VAF plus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Kent, England
- Age at study initiation: 7 to 10 weeks
- Weight at study initiation: 205 - 243 g
- Housing: individually in metal cages with wire mesh floors
- Diet: standard laboratory rodent diet (Biosure, LAD 1), ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 22
- Humidity (%): 46
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 3 Dec To: 17 Dec 1990

Type of coverage:

occlusive

Vehicle:

water

Details on dermal exposure:

TEST SITE
- Area of exposure: 5 x 5 cm clipped skin of the dorso-lumbar region
- % coverage: 10% of total body surface
- Type of wrap if used: gauze which was held in place by an impermeable dressing

REMOVAL OF TEST SUBSTANCE
- Washing: in warm water (30-40°C) and blotting dry with absorbent paper
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 3.0 mL/kg bw
- Concentration (if solution): 66.7% (w/v) in distilled water

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: animals were observed twice daily and weighing was carried out on day 1, 8 and 15 (day1 = day of dosing)
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, dermal irritation (treated areas of skin were observed daily for signs of irritation and assessed according to the Draize scoring system)

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortalities were observed during the study period.

Clinical signs:

other: No clinical sigsn of systemic effects were seen.

Gross pathology:

No abnormalitites were found at terminal necropsy.

Other findings:

- Other observations: No dermal reactions at the site of application were observed.

Table 1: Body weights

Sex	Dose	Animal number	Bodyweight in g (individual gain weekly) at
	(mg/kg)		Day1	Day8	Day15
Male	2000	1	243	320 (77)	384 (64)
		2	206	269 (63)	333 (64)
		3	232	310 (78)	378 (68)
		4	234	317 (83)	387 (70)
		5	231	306 (75)	377 (71)
	Mean		229	304	372
Female	2000	6	214	238 (24)	247 (9)
		7	225	259 (34)	276 (17)
		8	205	209 (4)	225 (16)
		9	217	233 (16)	260 (27)
		10	205	221 (16)	238 (17)
	Mean		213	232	249

Interpretation of results:

GHS criteria not met

Conclusions:

The LD50 was determined to be > 2000 mg/kg.

Executive summary:

The acute dermal toxicity of the test substance to rats was investigated according to EEC B.3 and following GLP principles. A limit test using a dose of 2000 mg/kg was conducted.

No mortality was observed in males and females.

The dermal LD50 value of the test substance was established to be > 2000 mg/kg body weight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.5, of Regulation (EC) No 1907/2006.

Additional information

Acute oral toxicity

An acute oral toxicity study (limit test) was performed with 4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline) (CAS 107934-68-9) in Sprague-Dawley rats, according to OECD 401 (Glaza, 1988a). 5 rats per sex received a single dose of 5000 mg/kg bw by gavage. There were no mortalities. Clinical signs that were reported included diarrhea observed in 1 male animal 4 hours after administration and miosis starting at 2.5 hours following administration until day 1 and day 2 in 2 male animals. Some female animals showed miosis (1, 2.5 and 4 hours after administration and on day 1), diarrhea (1, 2.5 and 4 hours post administration), hypoactivity and lacrimation (2.5 and 4 hours post administration) and hypersensitivity to touch (one animal 4 hours after administration). There was no effect on the body weight gain of the animals. Necropsy revealed multiple red pinpoint foci in the right salivary gland and diffusely dark red submandibular bilateral lymph nodes in one female animal. A second female had an enlarged pelvis in the right kidney. The findings in the two female rats were considered to be incidental and unrelated to exposure. All other animals were without visible lesions. The acute oral LD50 in rats was found to be > 5000 mg/kg bw.

Acute inhalation toxicity

The acute inhalation toxicity of 4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline) (CAS 107934-68-9) was assessed in a study performed according to OECD Guideline 436 in compliance with GLP (WIL Research Europe B.V., 2015). Three Wistar rats/sex were administered 3 ± 0.1 mg/L (actual concentration) of the test substance as an aerosol via nose-only exposure for 4 hours. The applied test concentration was the technically maximum attainable concentration. The nominal concentration was 44 mg/L. Concentration measurements were equally distributed over time and showed that it was difficult to maintain a stable test concentration at the technically maximum attainable concentration The generation was interrupted 4-times in order to remove test substance deposits from the system. The generation time was elongated to compensate for these interruptions. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (gsd) was determined to be 3.2 µm (gsd 2.1) and 3.9 µm (gsd 2.3) in two independent measurements during the exposure period.

No mortality occurred up to the end of the 14-day observation period. Rapid breathing was noted for the animals during exposure. No clinical signs were observed following the inhalative exposure. All females lost body weight during the first week up to Day 8. Two females regained weight during the second week resulting in an overall body weight gain during the 14-day observation period, while one female remained below the initial body weight at the start of the study. Overall body weight gain in male rats was considered normal. No abnormalities were reported during the macroscopic post mortem examination. Based on the results of the conducted study, the LC50 value for the test substance is considered to be > 3 mg/L in male and female rats.

Acute dermal toxicity

An acute dermal toxicity study (limit test) was performed with 4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline) (CAS 107934-68-9) in Crl. (CD) SD BR VAF plus rats, equivalent to OECD 402 (Baldrick, 1991). 2000 mg/kg bw of the test substance was applied to the clipped skin of 5 rats/sex/dose under an occlusive dressing for 24 hours. No mortality occurred and no clinical signs were observed. Slightly lower body weight gains were noted for one female on Day 8 and a second one on Day 15 compared to the others, but these rats achieved anticipated gains on Day 15 and Day 8, respectively. All other rats achieved anticipated body weight gains during the study period. No abnormalitites were found at terminal necropsy. No dermal reactions were observed at the site of application. The LD50 is considered to be > 2000 mg/kg bw.

Justification for selection of acute toxicity – oral endpoint
There is only one study available.

Justification for selection of acute toxicity – inhalation endpoint
There is only one study available.

Justification for selection of acute toxicity – dermal endpoint
There is only one study available.

Justification for classification or non-classification

The available data on acute oral, inhalation and dermal toxicity of 4,4´-(9H-fluoren-9-ylidene)bis(2-chloroaniline) does not meet the criteria for classification of Acute Toxicity according to Regulation (EC) 1272/2008 and is therefore conclusive but not sufficient for classification.