1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

other information

Study period:

From 25 SEPTEMBER 1996 to 17 OCTOBER 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study has been carried out under GLP and according to international OECD guidelines (OECD guideline 417).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Objective of study:

toxicokinetics

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

No control group and no (second) higher dose

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

EC Number:

214-946-9

EC Name:

1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

Cas Number:

1222-05-5

Molecular formula:

C18H26O

IUPAC Name:

4,6,6,7,8,8-hexamethyl-1H,3H,4H,6H,7H,8H-indeno[5,6-c]pyran

Details on test material:

- Name of test material (as cited in study report): HHCB
- Molecular formula (if other than submission substance): C18H26O
- Molecular weight (if other than submission substance): 258.41
- Physical state: Almost colourless viscous liquid
- Lot/batch No.: 960214 (radiolabelled test substance)
- Lot/batch No.: 7953-1 (non-radiolabelled test substance)
- Radiochemical purity (if radiolabelling): 98.7%
- Specific activity (if radiolabelling): 36.7 mCi/mmol; 142 uCi/mg; 5.255 MBq/mg
- Specific activity (diluted for dose administration): 8.78 mCi/mmol; 33.97 uCi/mg; 1.257 MBq/mg
- Locations of the label (if radiolabelling): In the centre of the aromatic ring
- Source of the radiolabelled test substance: Wizard Laboratories Inc., USA
- Source of the non-radiolabelled test substance: International Flavours and Fragrances , Union Beach, NJ, USA

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Uk, Ltd.
- Age at study initiation: ca. 10-11 weeks
- Weight at study initiation: 213-230 g
- Strain: Sprague-Dawley (Crl: CD®BR)
- Housing: Stainless steel cages with suspended mesh floors except for excretion/balance experiments, during which the rats were housed in glass Metabowls designed to facilitate the separation of urine and faeces and the trapping of expired air.
- Individual metabolism cages: yes
- Diet (ad libitum): LAD1, Special diet services Ltd.
- Water (ad libitum): Anglian Water mains supply
- Acclimation period: 5 days before administration of the dose

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C±2°C
- Humidity (%): 40-60%
- Air changes (per hr): 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intravenous

Vehicle:

other: Ethanol/Emulphor EL 620/isotonic saline (1:1:7) solution

Details on exposure:

TEST SITE
- Exposure: Intravenous injection into the tail vein

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 5 mL/kg
- concentration (if solution): 0.4 mg/mL

Duration and frequency of treatment / exposure:

The test substance was administrated as a single dose.

Doses / concentrations

Remarks:

Doses / Concentrations:
Rats received a injection in the tail vein of 2 mg/kg HHCB.

No. of animals per sex per dose / concentration:

In the preliminary experiment 4 female rats (2 mg/kg)
In the main experiment 52 female rats (2 mg/kg)

Control animals:

no

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, cage washes, liver, kidneys, carcasses
- Time and frequency of sampling: In the main study rats were sacrified in groups of 4 and 5, 15 and 30 min., and 1, 2, 4, 6, 12, 24, and 48 hours and, and 7 , 14 and 28 days. Urine and faeces were collected at 24-hour intervals from animals sacrified at 7 days. 14C-carbon dioxide was collected by passing expired air through 2 traps containing 2-ethoxyethanol/ethanolamine. Contents of the traps were collected at 24-hours intervals up to 28 hours. Cage washes were made with water at the time of sacrifice (168 hours).

Results and discussion

Preliminary studies:

In a preliminary experiment 4 rats were administered single intravenous doses of non-radiolabelled HHCB. No adverse effects were observed at this dose level (2 mg/kg).

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Mean concentrations of radioactivity in plasma declined from 2.57 jig equivalents/g at 5 minutes to 1.54, 1.31, 1.04, 0.564 and 0.249 jig equivalents/g at 30 minutes, 2, 6 12 and 24 hours respectively. Between 48 hours and 14 days radioactivity declined from 0.102 to 0.00199 jig equivalents/g with a half life of 2.1 days after which concentrations declined more slowly to 0.00050 jig equivalents/g at 28 days. Analysis of plasma samples for parent compound showed that 7.2 % and 1.6 % of plasma radioactivity were associated with parent HHCB at 2 and 24 hours respectively.
Mean concentrations of radioactivity in whole-blood declined from 1.58 jig equivalents/g at 5 minutes to 0.914, 0.716, 0.565, 0.332, 0.148 jig equivalents/g at 30 minutes, 2, 6 12 and 24 hours respectively. Concentrations declined to 0.0108 j.i.g equivalents/g at 7 days. Between 7 and 28 days radioactivity declined to 0.00185 ji.g equivalents/g with a half life of 8.5 days. At earlier times radioactivity in blood was primarily associated with plasma. From 7 days the major proportion of blood radioactivity was associated with the cells.

Details on distribution in tissues:

Mean concentrations of radioactivity in liver declined from 8.83 jig equivalents/g at 5 minutes to 2.32 ug equivalents/g at 4 hours. Mean concentrations of radioactivity rose to 2.39 ug equivalents/g at 6 hours and then declined to 1.09 and 0.548 ug equivalents/g at 24 and 48 hours respectively. Between 7 and 28 days mean concentrations declined from 0.121 to 0.0221 ug equivalents/g. Mean concentrations of radioactivity in kidneys declined from 4.65 ug equivalents/g at 5 minutes to 2.38, 1.26, 0.8 17 and 0.227 ug equivalents/g at 30 minutes, 2, 6 and 24 hours. Concentrations declined to 0.0237 ug equivalents/g at 7 days and to 0.004 15 ug equivalents/g at 28 days with a half life of 8.6 days. Mean concentrations of radioactivity in fat rose from 1.21 ug equivalents/g at 5 minutes to 6.64 ug equivalents/g at 2 hours after which concentrations declined to 4.20 ug equivalents/g at 6 hours and then rose to a second peak of 4.75 ug equivalents/g at 12 hours. Between 24 hours and 14 days radioactivity declined from 3.66 ug equivalents/g to 0.098 9 ug equivalents/g with a half life of 2.6 days after which concentrations declined more slowly to 0.0260 ug equivalents/g at 28 days. Analysis of fat samples for parent compound showed that most of the radioactivity (57 - 77%) was associated with HHCB. Concentrations of HHCB of 3.81, 2.45 and 0.442 ug/g were found in fat from animals sacrificed at 2 hours, 1 day and 7 days respectively.

Details on excretion:

In animals from which excreta were collected no radioactivity was detected in expired air (0 - 48 hours). A mean recovery of 91.8 % dose was recovered from other excreta and tissues. A mean of 28.1 % dose was detected in urine of which 23.3 % dose was excreted during 0 - 48 hours. In faeces a mean of 61.0 % dose was excreted of which 8.9 % was excreted during 0 - 24 hours, 25.6 % during 24 - 48 hours and 17.7 % during 48 - 72 hours. Mean recoveries of 0.01, 0.25 and 2.14 % dose respectively were found in kidneys, liver and remaining carcass.

Metabolite characterisation studies

Metabolites identified:

no

Any other information on results incl. tables

Recovery of radioactivity in excreta and tissues of rats after intravenous doses of 14C-HHCB at a dose of 2 mg/kg.

Time (hours)	Sacrifice time/animal number and sex
		168 hours
	41	42	43	44	Mean	SD
Tissues
Kidney	0.01	0.01	0.01	0.01	0.01	0.01
Liver	0.24	0.3	0.26	0.19	0.25	0.05
Carcass	1.67	3.07	2.33	1.48	2.14	0.72
Urine
0-24	19.4	16.65	12.73	21.47	17.56	3.78
24-48	4.33	6.41	6.87	5.42	5.76	1.13
48-72	1.58	3.74	3.02	1.7	2.51	1.05
72-96	0.72	1.77	1.32	0.79	1.15	0.49
96-120	0.46	0.78	0.64	0.37	0.56	0.18
120-144	0.22	0.53	0.4	0.24	0.335	0.15
144-168	0.16	0.38	0.29	0.18	0.25	0.1
Total urine	26.87	30.26	25.27	30.17	28.14	2.48
Faeces
0-24	16.65	0.27	0.17	18.68	8.98	10.11
24-48	32.23	22.6	19.98	27.63	25.61	5.44
48-72	7.48	21.56	34.35	7.42	17.7	12.94
72-96	4.21	6.06	5.43	3.14	4.71	1.3
96-120	1.68	2.11	2.53	1.37	1.92	0.51
120-144	0.84	1.78	2	0.73	1.34	0.65
144-168	0.53	1.06	1	0.56	0.79	0.28
Total faeces	63.62	55.44	65.46	59.53	61.01	4.47
Cage washings	0.15	0.17	0.33	0.14	0.2	0.09
Air traps
0-24	nd	nd	nd	nd	nd	nd
24-48	nd	nd	nd	nd	nd	nd
Total recovery	92.56	89.25	93.66	91.52	91.75	1.88

Concentations of radioactivity in tissues of rats after intravenous doses of 14C-HHCB at a dose level of 2 mg/kg.

		Tissues
Time	Fat	Kidney	Liver	Plasma	Whole-blood
5 min	1.21	4.65	8.83	2.57	1.58
15 min	1.84	3.11	5.9	1.94	1.17
30 min	3.29	2.38	4.73	1.54	0.914
1 hr	5.27	1.94	4.03	1.46	0.845
2 hrs	6.64	1.26	3	1.31	0.716
4 hrs	5.55	0.914	2.32	1.06	0.584
6 hrs	4.2	0.817	2.39	1.04	0.565
12 hrs	4.75	0.503	1.99	0.564	0.332
24 hrs	3.66	0.227	1.09	0.249	0.148
2 days	2.17	0.092	0.548	0.102	0.0644
7 days	0.575	0.0237	0.121	0.011	0.00108
14 days	0.0989	0.00985	0.0407	0.00199	0.00438
28 days	0.026	0.00415	0.0221	0.0005	0.00185

Mean concentrations of HHCB in tissues

Time	Fat	Plasma
2 hrs	3.81	0.094
1 day	2.45	0.004
7 days	0.442

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): low bioaccumulation potential based on study results
After intravenous administration of the carbon- 14 radiolabelled compound, HHCB was eliminated in urine (28.1 % dose) and faeces (61.0 % dose). At the 7 day sacrifice, 0.25 %, dose remained in the liver and 2.14 % in the carcass.
Measurement of HHCB in fat showed that from 57% (2 hours) to 77% (7 days) of the tissue radioactivity was associated with parent compound. In plasma the proportion was 7% or less. This indicated that HHCB was taken up and retained by fat after dosing but was rapidly metabolised on release from the fat.

Executive summary:

In the present GLP compliant study, groups of four female Sprague Dawley CD rats (bodyweight range 213-230 g – age 10-11 weeks) received a single intravenous administration of 2 mg/kg bw ¹⁴C-HHCB (uniformly labelled in the aromatic ring – radiochemical purity 99%) in a 0.4 mg/ml ethanol/Emulphor EL 620/isotonic saline (1:1:7) solution in the tail vein and were sacrificed at 5, 15, 30 min and 1, 2, 4, 6, 12, 24 and 48 hr and 7, 14 and 28 days. Tissues (fat, kidney, liver) were weighed and blood was collected by cardiac puncture. Urine, faeces and air were collected from the 4 animals that were sacrificed at day 7 after every 24 hrs until 168 hours (air up to 48 hrs). The recovery of radioactivity in these 4 animals represented 91.8 % of the dose administered: 89.3% in excreta plus cage washings, 2.14% in the carcass and 0.25% in the liver.

Maximum concentrations of radioactivity were observed in all tissues at 5 min (earliest time of measurement) except for the fat where the maximum was at 2 hr. Between 48 hr and 14 days, radioactivity in the plasma and fat decreased with apparent half-lives of elimination of 2.1 and 2.6 days respectively. In the fat, the majority of radioactivity (57-77%) was associated with parent HHCB. In whole blood, concentrations declined between 7 and 28 days with a half-life of 8.5 days with the majority of the radioactivity being associated with the cells while at earlier times it was primarily associated with the plasma. In the kidneys, the decline between 7 and 28 days was with a half-life of 8.6 days.

The majority of the radioactivity (53% of the dose via faeces and 23% of the dose via urine) was excreted during the first 72 hr or 48 hr post-dosing for faeces and urine, respectively. Over the entire collection period (168 hr), the excretion via these routes amounted to 61% and 28.1% for faeces and urine. Exhalation of radioactivity could not be detected.